Chronic obstructive pulmonary disease (COPD) biomarkers and uses thereof

ABSTRACT

Methods, compositions, and kits for predicting whether a subject with chronic obstructive pulmonary disease (COPD) or at risk of developing COPD is likely to have progressive COPD are provided. Methods, compositions, and kits for predicting whether lung function is likely to decline in a subject with COPD or at risk for developing COPD are also provided.

This application is a continuation of U.S. patent application Ser. No. 14/074,086, filed Nov. 7, 2013, now U.S. Pat. No. 9,103,837, which claims the benefit of U.S. Provisional Application No. 61/723,551, filed Nov. 7, 2012, each of which is incorporated by reference herein in its entirety for any purpose.

FIELD OF THE INVENTION

The present application relates generally to the detection of biomarkers and the characterization of chronic obstructive pulmonary disease (COPD), for example, to predict lung function decline in individuals COPD. In various embodiments, the invention relates to one or more biomarkers, methods, devices, reagents, systems, and kits for characterizing COPD in an individual.

BACKGROUND

The following description provides a summary of information and is not an admission that any of the information provided or publications referenced herein is prior art to the present application.

Chronic Obstructive Pulmonary Disease (COPD) is a lung disease often associated with a history of cigarette smoking in which there is progressive and irreversible obstruction of the airways as well as destruction of the alveoli. The prevalence of COPD in the US is 13.1 million people and it is the fourth leading cause of death with 126,005 deaths of persons aged ≧25 years in 2005. Approximately 12 million adults have undiagnosed COPD and emergency room visits and hospitalizations (700,000/year in US) due to exacerbation of COPD are a huge financial burden to health care. The total economic burden of COPD is almost $50Bn, with $30Bn of direct health care expenditures.

Patients with symptoms such as chronic cough, chronic sputum production, dyspnea, or a history of inhalation exposure to tobacco smoke, occupational dust may be diagnosed with COPD using a spirometer to measure pulmonary function (pulmonary function tests—PFTs). The most common measures of pulmonary function are the forced expiratory volume in one second (FEV1) and the forced vital capacity (FVC). Many people with COPD do not deteriorate, or deteriorate very slowly, whereas some people deteriorate rapidly. The latter group would benefit from more aggressive treatment of infections, glucocorticoid or bronchodilator therapy, and enrollment in aggressive smoking cessation programs.

Currently, there is no method of predicting the prognosis of COPD patients, for example, to discriminate between individuals with COPD whose lung function is likely to decline and individuals with COPD with more stable lung function.

SUMMARY

In some embodiments, methods of predicting whether lung function will decline in a subject with chronic obstructive pulmonary disease (COPD) are provided. In some embodiments, a method comprises detecting the level of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine biomarkers selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7 and optionally one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of IGFBP1 and optionally one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of CCL5 and optionally one or more of SIGLEC7, IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7 and IGFBP1 and optionally one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of IGFBP1 and CCL5 and optionally one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7 and CCL5 and optionally one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7, IGFBP1, and CCL5 and optionally one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, lung function is predicted to decline if the level of SIGLEC7, IGFBP1, IGFBP2, LYVE1, PIGR, ANGPT2, GSK3β, CCDC80, CHST15, COL18A1, CCL5, FST, LGALS3, or LGMN is higher than a control level of the respective biomarker. In some embodiments, lung function is predicted to decline if the level of GPC2, PSPN, LTA LTB, FGF19, THPO, MST1R, HS6ST1, or IL10 is lower than a control level of the respective biomarker.

In some embodiments, a method of predicting whether lung function will decline in a subject with chronic obstructive pulmonary disease (COPD) comprises forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 4, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject. In some embodiments, N is 1 to 30. In some embodiments, N is 2 to 30. In some embodiments, N is 3 to 30. In some embodiments, N is 4 to 30. In some embodiments, N is 5 to 30. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a method of predicting whether lung function will decline in a subject with chronic obstructive pulmonary disease (COPD) comprises forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 6, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, N is 1 to 5. In some embodiments, N is 2 to 5. In some embodiments, N is 3 to 5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a method of predicting whether lung function will decline in a subject with chronic obstructive pulmonary disease (COPD) comprises detecting the levels of a biomarker protein or set of biomarker proteins listed in Table 7, Table 8, or Table 9, and optionally one, two, or three or more additional markers from Table 6. In some embodiments, a set of biomarker proteins with a sensitivity+specificity value of 1.3 or greater, 1.35 or greater, 1.4 or greater, 1.45 or greater, 1.5 or greater, is selected from Table 8 or Table 9, and optionally one, two, or three or more additional markers from Table 6.

In some embodiments, methods of predicting whether lung function will decline in a subject at risk of developing COPD are provided. In some embodiments, a method comprises detecting the level of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine biomarkers selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7 and optionally one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of IGFBP1 and optionally one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of CCL5 and optionally one or more of SIGLEC7, IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7 and IGFBP1 and optionally one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of IGFBP1 and CCL5 and optionally one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7 and CCL5 and optionally one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7, IGFBP1, and CCL5 and optionally one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, lung function is predicted to decline if the level of SIGLEC7, IGFBP1, IGFBP2, LYVE1, PIGR, ANGPT2, GSK3β, CCDC80, CHST15, COL18A1, CCL5, FST, LGALS3, or LGMN is higher than a control level of the respective biomarker. In some embodiments, lung function is predicted to decline if the level of GPC2, PSPN, LTA LTB, FGF19, THPO, MST1R, HS6ST1, or IL10 is lower than a control level of the respective biomarker.

In some embodiments, a method of predicting whether lung function will decline in a subject at risk of developing COPD comprises forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 4, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject. In some embodiments, N is 1 to 30. In some embodiments, N is 2 to 30. In some embodiments, N is 3 to 30. In some embodiments, N is 4 to 30. In some embodiments, N is 5 to 30. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a method of predicting whether lung function will decline in a subject at risk of developing COPD comprises forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 6, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, N is 1 to 5. In some embodiments, N is 2 to 5. In some embodiments, N is 3 to 5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a method of predicting whether lung function will decline in a subject at risk of developing COPD comprises detecting the levels of a biomarker protein or set of biomarker proteins listed in Table 7, Table 8, or Table 9, and optionally one, two, or three or more additional markers from Table 6. In some embodiments, a set of biomarker proteins with a sensitivity+specificity value of 1.3 or greater, 1.35 or greater, 1.4 or greater, 1.45 or greater, 1.5 or greater, is selected from Table 8 or Table 9, and optionally one, two, or three or more additional markers from Table 6.

In some embodiments, methods of predicting whether COPD will progress in a subject with COPD are provided. In some embodiments, a method comprises detecting the level of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine biomarkers selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7 and optionally one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of IGFBP1 and optionally one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of CCL5 and optionally one or more of SIGLEC7, IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7 and IGFBP1 and optionally one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of IGFBP1 and CCL5 and optionally one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7 and CCL5 and optionally one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7, IGFBP1, and CCL5 and optionally one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, COPD is predicted to progress if the level of SIGLEC7, IGFBP1, IGFBP2, LYVE1, PIGR, ANGPT2, GSK3β, CCDC80, CHST15, COL18A1, CCL5, FST, LGALS3, or LGMN is higher than a control level of the respective biomarker. In some embodiments, COPD is predicted to progress if the level of GPC2, PSPN, LTA LTB, FGF19, THPO, MST1R, HS6ST1, or IL10 is lower than a control level of the respective biomarker.

In some embodiments, a method of predicting whether COPD will progress in a subject with COPD comprises forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 4, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject. In some embodiments, N is 1 to 30. In some embodiments, N is 2 to 30. In some embodiments, N is 3 to 30. In some embodiments, N is 4 to 30. In some embodiments, N is 5 to 30. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a method of predicting whether COPD will progress in a subject with COPD comprises forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 6, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, N is 1 to 5. In some embodiments, N is 2 to 5. In some embodiments, N is 3 to 5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a method of predicting whether COPD will progress in a subject with COPD comprises detecting the levels of a biomarker protein or set of biomarker proteins listed in Table 7, Table 8, or Table 9, and optionally one, two, or three or more additional markers from Table 6. In some embodiments, a set of biomarker proteins with a sensitivity+specificity value of 1.3 or greater, 1.35 or greater, 1.4 or greater, 1.45 or greater, 1.5 or greater, is selected from Table 8 or Table 9, and optionally one, two, or three or more additional markers from Table 6.

In some embodiments, methods of predicting whether a subject at risk of developing COPD will develop progressive COPD are provided. In some embodiments, a method comprises detecting the level of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine biomarkers selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7 and optionally one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of IGFBP1 and optionally one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of CCL5 and optionally one or more of SIGLEC7, IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7 and IGFBP1 and optionally one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of IGFBP1 and CCL5 and optionally one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7 and CCL5 and optionally one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, a method comprises detecting the level of SIGLEC7, IGFBP1, and CCL5 and optionally one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in a sample from the subject. In some embodiments, the subject is predicted to develop progressive COPD if the level of SIGLEC7, IGFBP1, IGFBP2, LYVE1, PIGR, ANGPT2, GSK3β, CCDC80, CHST15, COL18A1, CCL5, FST, LGALS3, or LGMN is higher than a control level of the respective biomarker. In some embodiments, the subject is predicted to develop progressive COPD if the level of GPC2, PSPN, LTA LTB, FGF19, THPO, MST1R, HS6ST1, or IL10 is lower than a control level of the respective biomarker.

In some embodiments, a method of predicting whether a subject at risk of developing COPD will develop progressive COPD comprises forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 4, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject. In some embodiments, N is 1 to 30. In some embodiments, N is 2 to 30. In some embodiments, N is 3 to 30. In some embodiments, N is 4 to 30. In some embodiments, N is 5 to 30. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a method of predicting whether a subject at risk of developing COPD will develop progressive COPD comprises forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 6, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, N is 1 to 5. In some embodiments, N is 2 to 5. In some embodiments, N is 3 to 5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a method of predicting whether a subject at risk of developing COPD will develop progressive COPD comprises detecting the levels of a biomarker protein or set of biomarker proteins listed in Table 7, Table 8, or Table 9, and optionally one, two, or three or more additional markers from Table 6. In some embodiments, a set of biomarker proteins with a sensitivity+ specificity value of 1.3 or greater, 1.35 or greater, 1.4 or greater, 1.45 or greater, 1.5 or greater, is selected from Table 8 or Table 9, and optionally one, two, or three or more additional markers from Table 6.

In any of the embodiments described herein, the method may comprise detecting the level of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight biomarkers selected from IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, and IGFBP4 in a sample from the subject. In some embodiments, lung function is predicted to decline or COPD is predicted to progress if the level of IGFBP2, HSPA1A, SLPI, SOD2, CCL22, or IGFBP4 is higher than a control level of the respective biomarker. In some embodiments, lung function is predicted to decline or COPD is predicted to progress if the level of CXCL5 or FGFR2 is lower than a control level of the respective protein.

In any of the embodiments described herein, the method may comprise detecting at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine biomarkers selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject. In any of the embodiments described herein, the method may comprise detecting SIGLEC7, and optionally one or more of IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject. In any of the embodiments described herein, the method may comprise detecting IGFBP1, and optionally one or more of SIGLEC7, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject. In any of the embodiments described herein, the method may comprise detecting CCL5 and optionally one or more of IGFBP1, SIGLEC7, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject. In any of the embodiments described herein, the method may comprise detecting SIGLEC7 and CCL5, and optionally one or more of IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject. In any of the embodiments described herein, the method may comprise detecting SIGLEC7 and IGFBP1, and optionally one or more of GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject. In any of the embodiments described herein, the method may comprise detecting SIGLEC7, IGFBP1, and CCL5, and optionally one or more of GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject. In any of the embodiments described herein, the method may comprise detecting IGFBP1 and CCL5, and optionally one or more of SIGLEC7, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject.

In any of the embodiments described herein, the method may comprise detecting the level of at least one, at least two, at least three, at least four, at least five, at least six, or seven biomarkers selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, and IGFBP-7 in a sample from the subject. In any of the embodiments described herein, the method may comprise detecting the level of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine biomarker selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, CCL5, GSK3β, THPO, GPC2, ANGPT2, PIGR, and LGMN. In any of the embodiments described herein, the method may comprise detecting the level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine biomarkers selected from SIGLEC7, IGFBP1, IGFBP2, PSPN, LTA LTB, CCL5, GSK3β, THPO, GPC2, ANGPT2, PIGR, and LGMN. In any of the embodiments described herein, the method may comprise detecting the level of at least one, at least two, at least three, or four biomarkers selected from SIGLEC7, IGFBP1, PSPN, and GPC2. In any of the embodiments described herein, the method may comprise detecting SIGLEC7 and CCL5. In any of the embodiments described herein, the method may comprise detecting the level of at least one, at least two, or three biomarkers selected from SIGLEC7, CCL5, and IGFBP1. In any of the embodiments described herein, the method may comprise detecting the levels of at least two, at least three, at least four, at least five, at least six, or seven biomarkers selected from SIGLEC7, CCL5, IGFBP2, LTA LTB, PSPN, GSK3β, and IGFBP1. In any of the embodiments described herein, the method may comprise detecting SIGLEC7, IGFBP2, CCL5, IGFBP1, LTA LTB, and PSPN.

In any of the embodiments described herein, the each biomarker may be a protein biomarker. In any of the embodiments described herein, the method may comprise contacting biomarkers of the sample from the subject with a set of biomarker capture reagents, wherein each biomarker capture reagent of the set of biomarker capture reagents specifically binds to a biomarker being detected. In some embodiments, each biomarker capture reagent of the set of biomarker capture reagents specifically binds to a different biomarker being detected. In any of the embodiments described herein, each biomarker capture reagent may be an antibody or an aptamer. In any of the embodiments described herein, each biomarker capture reagent may be an aptamer. In any of the embodiments described herein, at least one aptamer may be a slow off-rate aptamer. In any of the embodiments described herein, at least one slow off-rate aptamer may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 nucleotides with modifications. In some embodiments, the modifications are hydrophobic modifications. In some embodiments, the modifications are hydrophobic base modifications. In some embodiments, one or more of the modifications may be selected from the modifications shown in FIG. 12. In some embodiments, each slow off-rate aptamer binds to its target protein with an off rate (t_(1/2)) of ≧30 minutes, ≧60 minutes, ≧90 minutes, ≧120 minutes, ≧150 minutes, ≧180 minutes, ≧210 minutes, or ≧240 minutes.

In any of the embodiments described herein, the sample may be a blood sample. In some embodiments, the blood sample is selected from a serum sample and a plasma sample.

In any of the embodiments described herein, a method may further comprise treating the subject for COPD. In some embodiments, treating the subject for COPD comprises at least one treatment selected from a smoking cessation program, a long-acting bronchodilator, an inhaled corticosteroid, and a systemic corticosteroid. In some embodiments, the smoking cessation program comprises a therapeutic agent that aids in smoking cessation, behavior modification therapy, or both. In some embodiments, the long-acting bronchodilator is selected from a β2-agonist and an anticholinergic. In some embodiments, the β2-agonist is selected from salmeterol, formoterol, bambuterol, and indacaterol; and the anticholinergic is selected from tiotropium and ipratropium bromide. In some embodiments, the inhaled corticosteroid is selected from beclomethasone, budesonide, flunisolide, fluticasone, mometasone, and triamcinolone. In some embodiments, the systemic corticosteroid is selected from methylprednisolone, prednisolone, and prednisone.

In some embodiments, methods of monitoring progression of COPD and/or monitoring declining lung function in a subject are provided. In some embodiments, a method comprises detecting the level of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine biomarkers selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject at a first time point. In some embodiments, the method further comprises measuring the level of the at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine biomarkers at a second time point. In some embodiments, a method comprises detecting the level of SIGLEC7 and optionally one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, a method comprises detecting the level of IGFBP1 and optionally one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, a method comprises detecting the level of CCL5 and optionally one or more of SIGLEC7, IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, a method comprises detecting the level of SIGLEC7 and IGFBP1 and optionally one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, a method comprises detecting the level of IGFBP1 and CCL5 and optionally one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, a method comprises detecting the level of SIGLEC7 and CCL5 and optionally one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, a method comprises detecting the level of SIGLEC7, IGFBP1, and CCL5 and optionally one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, COPD is progressing and/or lung function is declining if the level of SIGLEC7, IGFBP1, IGFBP2, LYVE1, PIGR, ANGPT2, GSK3β, CCDC80, CHST15, COL18A1, CCL5, FST, LGALS3, or LGMN is higher at the second time point than at the first time point. In some embodiments, COPD is progressing and/or lung function is declining if the level of GPC2, PSPN, LTA LTB, FGF19, THPO, MST1R, HS6ST1, or IL10 is lower at the second time point than at the first time point. In some embodiments, a method further comprises detecting the level of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight biomarkers selected from IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, and IGFBP4 in the sample from the subject. In some embodiments, COPD is progressing and/or lung function is declining if the level of IGFBP2, HSPA1A, SLPI, SOD2, CCL22, or IGFBP4 is higher at the second time point than at the first time point. In some embodiments, COPD is progressing and/or lung function is declining if the level of CXCL5 or FGFR2 is lower at the second time point than at the first time point.

In some embodiments, a method of monitoring progression of COPD and/or monitoring declining lung function comprises forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 4, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject. In some embodiments, N is 1 to 30. In some embodiments, N is 2 to 30. In some embodiments, N is 3 to 30. In some embodiments, N is 4 to 30. In some embodiments, N is 5 to 30. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a method of monitoring progression of COPD and/or monitoring declining lung function comprises forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 6, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, N is 1 to 5. In some embodiments, N is 2 to 5. In some embodiments, N is 3 to 5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a method of monitoring progression of COPD and/or monitoring declining lung function comprises detecting the levels of a biomarker protein or set of biomarker proteins listed in Table 7, Table 8, or Table 9, and optionally one, two, or three or more additional markers from Table 6. In some embodiments, a set of biomarker proteins with a sensitivity+specificity value of 1.3 or greater, 1.35 or greater, 1.4 or greater, 1.45 or greater, 1.5 or greater, is selected from Table 8 or Table 9, and optionally one, two, or three or more additional markers from Table 6.

In some embodiments, a method of monitoring treatment of COPD in a subject is provided, comprising detecting the level of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine biomarkers selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from the subject at a first time point. In some embodiments, the method further comprises measuring the level of the at least one biomarker at a second time point. In some embodiments, a method comprises detecting the level of SIGLEC7 and optionally one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, a method comprises detecting the level of IGFBP1 and optionally one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, a method comprises detecting the level of CCL5 and optionally one or more of SIGLEC7, IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, a method comprises detecting the level of SIGLEC7 and IGFBP1 and optionally one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, a method comprises detecting the level of IGFBP1 and CCL5 and optionally one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, a method comprises detecting the level of SIGLEC7 and CCL5 and optionally one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, a method comprises detecting the level of SIGLEC7, IGFBP1, and CCL5 and optionally one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN, in samples from the subject at the first and second time points. In some embodiments, the treatment is effective if the level of SIGLEC7, IGFBP1, IGFBP2, LYVE1, PIGR, ANGPT2, GSK3β, CCDC80, CHST15, COL18A1, CCL5, FST, LGALS3, or LGMN is lower at the second time point than at the first time point. In some embodiments, the treatment is effective if the level of GPC2, PSPN, LTA LTB, FGF19, THPO, MST1R, HS6ST1, or IL10 is higher at the second time point than at the first time point. In some embodiments, the method further comprises detecting the level of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight biomarkers selected from IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, and IGFBP4 in the sample from the subject. In some embodiments, COPD is progressing and/or lung function is declining if the level of IGFBP2, HSPA1A, SLPI, SOD2, CCL22, or IGFBP4 is higher at the second time point than at the first time point. In some embodiments, COPD is progressing and/or lung function is declining if the level of CXCL5 or FGFR2 is lower at the second time point than at the first time point.

In some embodiments, a method of monitoring treatment of COPD comprises forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 4, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject. In some embodiments, N is 1 to 30. In some embodiments, N is 2 to 30. In some embodiments, N is 3 to 30. In some embodiments, N is 4 to 30. In some embodiments, N is 5 to 30. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a method of monitoring treatment of COPD comprises forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 6, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, N is 1 to 5. In some embodiments, N is 2 to 5. In some embodiments, N is 3 to 5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a method of monitoring treatment of COPD comprises detecting the levels of a biomarker protein or set of biomarker proteins listed in Table 7, Table 8, or Table 9, and optionally one, two, or three or more additional markers from Table 6. In some embodiments, a set of biomarker proteins with a sensitivity+specificity value of 1.3 or greater, 1.35 or greater, 1.4 or greater, 1.45 or greater, 1.5 or greater, is selected from Table 8 or Table 9, and optionally one, two, or three or more additional markers from Table 6.

In some embodiments, kits are provided. In some embodiments, a kit comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine aptamers, wherein each aptamer specifically binds to a different target protein selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a kit comprises an aptamer that specifically binds SIGLEC7 and optionally one or more aptamers that specifically bind one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a kit comprises an aptamer that specifically binds IGFBP1 and optionally one or more aptamers that specifically bind one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a kit comprises an aptamer that specifically binds CCL5 and optionally one or more aptamers that specifically bind one or more of SIGLEC7, IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a kit comprises an aptamer that specifically binds SIGLEC7 and an aptamer that specifically binds IGFBP1 and optionally one or more aptamers that specifically bind one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a kit comprises an aptamer that specifically binds IGFBP1 and an aptamer that specifically binds CCL5 and optionally one or more aptamers that specifically bind one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a kit comprises an aptamer that specifically binds SIGLEC7 and an aptamer that specifically binds CCL5 and optionally one or more aptamers that specifically bind one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a kit comprises an aptamer that specifically binds SIGLEC7, an aptamer that specifically binds IGFBP1, and an aptamer that specifically binds CCL5 and optionally one or more aptamers that specifically bind one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, each aptamer binds to a different target protein.

In some embodiments, a kit comprises N aptamers, wherein each aptamer specifically binds to a biomarker protein listed in Table 4. In some embodiments, N is 1 to 30. In some embodiments, N is 2 to 30. In some embodiments, N is 3 to 30. In some embodiments, N is 4 to 30. In some embodiments, N is 5 to 30. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a kit comprises N aptamers, wherein each aptamer specifically binds to a biomarker protein listed in Table 6. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, N is 1 to 5. In some embodiments, N is 2 to 5. In some embodiments, N is 3 to 5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a kit comprises aptamers for detecting a biomarker protein or set of biomarker proteins listed in Table 7, Table 8, or Table 9, and optionally one, two, or three or more additional markers from Table 6. In some embodiments, a set of biomarker proteins with a sensitivity +specificity value of 1.3 or greater, 1.35 or greater, 1.4 or greater, 1.45 or greater, 1.5 or greater, is selected from Table 8 or Table 9, and optionally one, two, or three or more additional markers from Table 6.

In some embodiments, the kit comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine aptamers, wherein each aptamer specifically binds a different target protein selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, the kit comprises at least one, at least two, at least three, at least four, at least five, at least six, or seven aptamers, wherein each aptamer specifically binds a different target protein selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, and IGFBP-7. In some embodiments, the kit comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine aptamers, wherein each aptamer specifically binds to a different target protein selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, the kit comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine aptamers, wherein each aptamer specifically binds a different target protein selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, CCL5, GSK3β, THPO, GPC2, ANGPT2, PIGR, and LGMN. In some embodiments, the kit comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine aptamers, wherein each aptamer specifically binds a different target protein selected from SIGLEC7, IGFBP1, IGFBP2, PSPN, LTA LTB, CCL5, GSK3β, THPO, GPC2, ANGPT2, PIGR, and LGMN. In some embodiments, the kit comprises at least one, at least two, at least three, or four aptamers, wherein each aptamer specifically binds a different target protein selected from SIGLEC7, IGFBP1, PSPN, and GPC2. In some embodiments, the kit comprises an aptamer that specifically binds SIGLEC7 and an aptamer that specifically binds CCL5. In some embodiments, the kit comprises at least one, at least two, or three aptamers, wherein each aptamer specifically binds a different target protein selected from SIGLEC7, CCL5, and IGFBP1. In some embodiments, the kit comprises at least two, at least three, at least four, at least five, at least six, or seven aptamers, wherein each aptamer specifically binds a different target protein selected from SIGLEC7, CCL5, IGFBP2, LTA LTB, PSPN, GSK3β, and IGFBP1. In some embodiments, the kit comprises at least six aptamers, including a first aptamer that specifically binds SIGLEC7, a second aptamer that specifically binds IGFBP2, a third aptamer that specifically binds CCL5, a fourth aptamer that specifically binds IGFBP1, a fifth aptamer that specifically binds LTA LTB, and a sixth aptamer that specifically binds PSPN. In some embodiments, the kit comprises at least one aptamer that does not bind to a target protein selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, and IGFBP4.

In some embodiments, compositions are provided comprising proteins of a sample from a subject and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or nine aptamers, wherein each aptamer specifically binds to a different target protein selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a composition comprises proteins of a sample from a subject and an aptamer that specifically binds SIGLEC7 and optionally one or more aptamers that specifically bind one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a composition comprises proteins of a sample from a subject and an aptamer that specifically binds IGFBP1 and optionally one or more aptamers that specifically bind one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a composition comprises proteins of a sample from a subject and an aptamer that specifically binds CCL5 and optionally one or more aptamers that specifically bind one or more of SIGLEC7, IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a composition comprises proteins of a sample from a subject and an aptamer that specifically binds SIGLEC7 and an aptamer that specifically binds IGFBP1 and optionally one or more aptamers that specifically bind one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a composition comprises proteins of a sample from a subject and an aptamer that specifically binds IGFBP1 and an aptamer that specifically binds CCL5 and optionally one or more aptamers that specifically bind one or more of SIGLEC7, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a composition comprises proteins of a sample from a subject and an aptamer that specifically binds SIGLEC7 and an aptamer that specifically binds CCL5 and optionally one or more aptamers that specifically bind one or more of IGFBP1, IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a composition comprises proteins of a sample from a subject and an aptamer that specifically binds SIGLEC7, an aptamer that specifically binds IGFBP1, and an aptamer that specifically binds CCL5 and optionally one or more aptamers that specifically bind one or more of IGFBP2, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN

In some embodiments, a composition comprises proteins of a sample from a subject and N aptamers, wherein each aptamer specifically binds to a biomarker protein listed in Table 4. In some embodiments, N is 1 to 30. In some embodiments, N is 2 to 30. In some embodiments, N is 3 to 30. In some embodiments, N is 4 to 30. In some embodiments, N is 5 to 30. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a composition comprises proteins of a sample from a subject and N aptamers, wherein each aptamer specifically binds to a biomarker protein listed in Table 6. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, N is 1 to 5. In some embodiments, N is 2 to 5. In some embodiments, N is 3 to 5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, and CCL5. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7, IGFBP1, and PSPN. In some embodiments, at least one of the N biomarker proteins is selected from SIGLEC7 and IGFBP1.

In some embodiments, a composition comprises proteins of a sample from a subject and aptamers for detecting a biomarker protein or set of biomarker proteins listed in Table 7, Table 8, or Table 9, and optionally one, two, or three or more additional markers from Table 6. In some embodiments, a set of biomarker proteins with a sensitivity+specificity value of 1.3 or greater, 1.35 or greater, 1.4 or greater, 1.45 or greater, 1.5 or greater, is selected from Table 8 or Table 9, and optionally one, two, or three or more additional markers from Table 6.

In some embodiments, the composition comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or nine aptamers, wherein each aptamer specifically binds to a different target protein selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, the composition comprises at least one, at least two, at least three, at least four, at least five, at least six, or seven aptamers, wherein each aptamer specifically binds to a different target protein selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, and IGFBP-7. In some embodiments, the composition comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine aptamers, wherein each aptamer specifically binds to a different target protein selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, the composition comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or nine aptamers, wherein each aptamer specifically binds to a different target protein selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, CCL5, GSK3β, THPO, GPC2, ANGPT2, PIGR, and LGMN. In some embodiments, the composition comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or nine aptamers, wherein each aptamer specifically binds to a different target protein selected from SIGLEC7, IGFBP1, IGFBP2, PSPN, LTA LTB, CCL5, GSK3β, THPO, GPC2, ANGPT2, PIGR, and LGMN. In some embodiments, the composition comprises at least one, at least two, at least three, or four aptamers, wherein each aptamer specifically binds a different target protein selected from SIGLEC7, IGFBP1, PSPN, and GPC2. In some embodiments, the composition comprises an aptamer that specifically binds SIGLEC7 and an aptamer that specifically binds CCL5. In some embodiments, the composition comprises at least one, at least two, or three aptamers, wherein each aptamer specifically binds a different target protein selected from SIGLEC7, CCL5, and IGFBP1. In some embodiments, the composition comprises at least two, at least three, at least four, at least five, at least six, or seven aptamers, wherein each aptamer specifically binds a target protein selected from SIGLEC7, CCL5, IGFBP2, LTA LTB, PSPN, GSK3β, and IGFBP1. In some embodiments, the composition comprises at least six aptamers, including a first aptamer that specifically binds SIGLEC7, a second aptamer that specifically binds IGFBP2, a third aptamer that specifically binds CCL5, a fourth aptamer that specifically binds IGFBP1, a fifth aptamer that specifically binds LTA LTB, and a sixth aptamer that specifically binds PSPN. In some embodiments, the composition comprises at least one aptamer that does not bind to a target protein selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN.

In any of the embodiments, described herein, the sample in a composition may be a blood sample. In some embodiments, the blood sample is selected from a serum sample and a plasma sample.

In any of the embodiments described herein, a kit or composition may comprise at least one aptamer that is a slow off-rate aptamer. In any of the embodiments described herein, each aptamer of a kit or composition may be a slow off-rate aptamer. In some embodiments, at least one slow off-rate aptamer comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 nucleotides with modifications. In some embodiments, at least one nucleotide with a modification is a nucleotide with a hydrophobic base modification. In some embodiments, each nucleotide with a modification is a nucleotide with a hydrophobic base modification. In some embodiments, each hydrophobic base modification is independently selected from the modification in FIG. 12. In some embodiments, each slow off-rate aptamer in a kit binds to its target protein with an off rate (t_(1/2)) of ≧30 minutes, ≧60 minutes, ≧90 minutes, ≧120 minutes, ≧150 minutes, ≧180 minutes, ≧210 minutes, or ≧240 minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chart of the study subjects' FEV1% over time (in days), as described in Example 1. Each dot represents a time point at which a blood sample was taken from the patient.

FIG. 2 shows ROC plots for biomarker sets of various sizes, selected through a random forest model, as described in Example 2.

FIG. 3 shows box plots for each biomarker in Table 4 in the progressor and non-progressor study subjects, as described in Example 3.

FIG. 4 shows five ROC plots from the five-fold validation of the exemplary six-marker classifier and one ROC plot showing the pooled votes from all of the samples, as described in Example 3.

FIG. 5 shows an overlay of the ROC plot for the six biomarker set from FIG. 2 and the ROC plot for the pooled votes from all of the cross-validation sets from FIG. 4, as described in Example 3.

FIG. 6 shows ROC plots for the six biomarker set for each sample collection time point (index 1=TP1, index 2=TP2, etc.), as described in Example 4.

FIG. 7 shows box plots of the (left panel) non-progressor and progressor scores for all time points in study subjects diagnosed with COPD at baseline, and (right panel) prediction of whether the non-COPD subjects at baseline would progress or not progress, as described in Example 5.

FIG. 8 shows a ROC plot with an overlay of the six-marker classifier scores at baseline (TP1) and the six-marker classifier scores averaged over all time points, as described in Example 6.

FIG. 9 shows a series of ROC plots based on averages of increasing numbers of time points for each individual, as described in Example 6.

FIG. 10 illustrates a nonlimiting exemplary computer system for use with various computer-implemented methods described herein.

FIG. 11 illustrates a nonlimiting exemplary aptamer assay that can be used to detect one or more biomarkers in a biological sample.

FIG. 12 shows certain exemplary modified pyrimidines that may be incorporated into aptamers, such as slow off-rate aptamers.

FIG. 13A-C shows paired histograms showing a single marker naive Bayes classifiers (n=1; A), pairwise marker classifiers (n=2; B), and triplicate marker classifiers (n=3; C), as described in Example 8.

DETAILED DESCRIPTION

While the invention will be described in conjunction with certain representative embodiments, it will be understood that the invention is defined by the claims, and is not limited to those embodiments.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein may be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

Unless defined otherwise, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the invention, certain methods, devices, and materials are described herein.

All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.

As used in this application, including the appended claims, the singular forms “a,” “an,” and “the” include the plural, unless the context clearly dictates otherwise, and may be used interchangeably with “at least one” and “one or more.” Thus, reference to “an aptamer” includes mixtures of aptamers, reference to “a probe” includes mixtures of probes, and the like.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “contains,” “containing,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements may include other elements not expressly listed.

The present application includes biomarkers, methods, devices, reagents, systems, and kits for determining COPD prognosis.

In one aspect, one or more biomarkers are provided for use either alone or in various combinations to identify progressive COPD and/or predict whether lung function will decline, in an individual with COPD or at risk of developing COPD. As described in detail below, exemplary embodiments include the biomarkers provided in Table 4, which were identified using a multiplex aptamer-based assay.

Table 4 lists 30 biomarkers that are useful for distinguishing samples obtained from individuals with progressive COPD from samples from individuals with non-progressive COPD. The biomarkers in Table 4 are also useful for predicting whether lung function will decline in individuals with COPD or at risk of developing COPD.

While the described biomarkers are useful alone for providing a COPD prognosis, methods are also described herein for grouping the biomarkers with additional biomarkers from Table 4 and/or with additional biomarkers not listed in Table 4. In some embodiments, panels of at least two, at least three, at least four, at least five, or at least 6 biomarkers from Table 4 (with or without additional biomarkers not listed in Table 4) are provided. Certain nonlimiting exemplary panels are shown in Table 3.

Table 6 lists 16 biomarkers that are useful for distinguishing samples obtained from individuals with progressive COPD from samples from individuals with non-progressive COPD. The biomarkers in Table 6 are also useful for predicting whether lung function will decline in individuals with COPD or at risk of developing COPD.

While the described biomarkers are useful alone for providing a COPD prognosis, methods are also described herein for grouping the biomarkers with additional biomarkers from Table 6 and/or with additional biomarkers not listed in Table 6. In some embodiments, panels of at least two, at least three, at least four, at least five, or at least 6 biomarkers from Table 6 (with or without additional biomarkers not listed in Table 6) are provided. Certain nonlimiting exemplary panels are shown in Tables 7 and 8.

In some embodiments, the number and identity of biomarkers in a panel are selected based on the sensitivity and specificity for the particular combination of biomarker values. The terms “sensitivity” and “specificity” are used herein with respect to the ability to correctly classify an individual, based on one or more biomarker levels detected in a biological sample, as having progressive COPD or not having progressive COPD. “Sensitivity” indicates the performance of the biomarker(s) with respect to correctly classifying individuals with progressive COPD. “Specificity” indicates the performance of the biomarker(s) with respect to correctly classifying individuals who do not have progressive COPD. For example, 85% specificity and 90% sensitivity for a panel of markers used to test a set of control samples (such as samples from healthy individuals or subjects known to have non-progressive COPD) and test samples (such as samples from individuals with progressive COPD) indicates that 85% of the control samples were correctly classified as control samples by the panel, and 90% of the test samples were correctly classified as test samples by the panel.

In some embodiments, overall performance of a panel of one or more biomarkers is represented by the area-under-the-curve (AUC) value. The AUC value is derived from receiver operating characteristic (ROC) plots, which are exemplified herein. The ROC curve is the plot of the true positive rate (sensitivity) of a test against the false positive rate (1-specificity) of the test. The term “area under the curve” or “AUC” refers to the area under the curve of a receiver operating characteristic (ROC) curve, both of which are well known in the art. AUC measures are useful for comparing the accuracy of a classifier across the complete data range. Classifiers with a greater AUC have a greater capacity to classify unknowns correctly between two groups of interest (e.g., progressive COPD and non-progressive COPD). ROC curves are useful for plotting the performance of a particular feature (e.g., any of the biomarkers described herein and/or any item of additional biomedical information) in distinguishing between two populations (e.g., cases having progressive COPD and controls, which may be cases with non-progressive COPD). Typically, the feature data across the entire population (e.g., all COPD cases) are sorted in ascending order based on the value of a single feature. Then, for each value for that feature, the true positive and false positive rates for the data are calculated. The true positive rate is determined by counting the number of cases above the value for that feature and then dividing by the total number of cases. The false positive rate is determined by counting the number of controls above the value for that feature and then dividing by the total number of controls. Although this definition refers to scenarios in which a feature is elevated in cases compared to controls, this definition also applies to scenarios in which a feature is lower in cases compared to the controls (in such a scenario, samples below the value for that feature would be counted). ROC curves can be generated for a single feature as well as for other single outputs, for example, a combination of two or more features can be mathematically combined (e.g., added, subtracted, multiplied, etc.) to provide a single sum value, and this single sum value can be plotted in a ROC curve. Additionally, any combination of multiple features, in which the combination derives a single output value, can be plotted in a ROC curve.

In some embodiments, a method comprises detecting the level of at least one biomarker listed in Table 4 in a sample from a subject for determining whether a subject with COPD is likely to progress and/or whether lung function in a subject with COPD or at risk of developing COPD is likely to decline. In some such embodiments, the method comprises contacting the sample or a portion of the sample from the subject with at least one capture reagent, wherein each capture reagent specifically binds a biomarker whose levels are being detected. In some embodiments, the method comprises contacting the sample, or proteins from the sample, with at least one aptamer, wherein each aptamer specifically binds a biomarker whose levels are being detected.

In some embodiments, a method comprises detecting the level of at least one biomarker listed in Table 6 in a sample from a subject for determining whether a subject with COPD is likely to progress and/or whether lung function in a subject with COPD or at risk of developing COPD is likely to decline. In some such embodiments, the method comprises contacting the sample or a portion of the sample from the subject with at least one capture reagent, wherein each capture reagent specifically binds a biomarker whose levels are being detected. In some embodiments, the method comprises contacting the sample, or proteins from the sample, with at least one aptamer, wherein each aptamer specifically binds a biomarker whose levels are being detected.

In some embodiments, a method comprises detecting the level of at least one biomarker selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1 FST, HS6ST1, LGALS3, IL10, and LGMN in a sample from a subject with COPD or at risk of developing COPD, wherein if the level of SIGLEC7, IGFBP1, LYVE1, PIGR, ANGPT2, GSK3β, CCDC80, CHST15, COL18A1, CCL5, FST, LGALS3, or LGMN is higher than a control level of the respective biomarker, and/or if the level of GPC2, PSPN, LTA LTB, FGF19, THPO, MST1R, HS6ST1, or IL10 is lower than a control level of the respective biomarker, the subject is predicted, or likely, to have progressive COPD and/or lung function in the subject is predicted, or likely, to decline. In some embodiments, the method further comprises detecting the level of at least one biomarker selected from IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, and IGFBP4 in a sample from the subject, wherein if the level of IGFBP2, HSPA1A, SLPI, SOD2, CCL22, or IGFBP4 is higher than a control level of the respective biomarker, and/or if the level of CXCL5 or FGFR2 is lower than a control level of the respective protein, the subject is predicted, or likely, to have progressive COPD and/or lung function in the subject is predicted, or likely, to decline.

In some embodiments, a method comprises detecting at least one biomarker, at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, at least seven biomarkers, at least eight biomarkers, or at least nine biomarkers selected from the biomarkers in Table 4. In some embodiments, a method comprises detecting at least one biomarker, at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, at least seven biomarkers, at least eight biomarkers, or at least nine biomarkers selected from the biomarkers in Table 6. In some embodiments, a method comprises detecting at least one biomarker, at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, at least seven biomarkers, at least eight biomarkers, or at least nine biomarkers selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a method comprises detecting at least one biomarker, at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, at least seven biomarkers, at least eight biomarkers, or at least nine biomarkers selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, CCL5, THPO, MST1R, CHST15, COL18A1, FST, HS6ST1, LGALS3, IL10, and LGMN and detecting at least one biomarker, at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, at least seven biomarkers, or eight biomarkers selected from IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, and IGFBP4. In some embodiments, a method comprises detecting the levels of a biomarker protein or set of biomarker proteins listed in Table 7, Table 8, or Table 9 and optionally one or more additional markers from Table 6. In some embodiments, a set of biomarker proteins with a sensitivity+specificity value of 1.3 or greater, 1.35 or greater, 1.4 or greater, 1.45 or greater, 1.5 or greater, is selected from Table 8 or Table 9, and optionally one, two, or three or more additional markers from Table 6.

The biomarkers identified herein provide a number of choices for subsets or panels of biomarkers that can be used to effectively identify progressive COPD. Selection of the appropriate number of such biomarkers may depend on the specific combination of biomarkers chosen. In addition, in any of the methods described herein, except where explicitly indicated, a panel of biomarkers may comprise additional biomarkers not shown in Table 4. In some embodiments, a method comprises detecting the level of at least one biomarker, at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or seven biomarkers selected from SIGLEC7, IGFBP1, GPC2, LYVE1, PSPN, CCDC80, and IGFBP-7 in a sample from the subject. In some embodiments, a method comprises detecting the level of at least one biomarker, at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, at least seven biomarkers, at least eight biomarkers, or at least nine biomarkers selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, CCL5, GSK3β, THPO, GPC2, ANGPT2, and PIGR. In some embodiments, a method comprises detecting the level of at least one biomarker, at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, at least seven biomarkers, at least eight biomarkers, or at least nine biomarkers selected from SIGLEC7, IGFBP1, PSPN, LTA LTB, CCL5, GSK3β, THPO, GPC2, ANGPT2, PIGR, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a method comprises detecting the level of at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, at least seven biomarkers, at least eight biomarkers, or at least nine biomarkers selected from SIGLEC7, IGFBP1, IGFBP2, PSPN, LTA LTB, CCL5, GSK3β, THPO, GPC2, ANGPT2, and PIGR. In some embodiments, a method comprises detecting the level of at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, at least seven biomarkers, at least eight biomarkers, or at least nine biomarkers selected from SIGLEC7, IGFBP1, IGFBP2, PSPN, LTA LTB, CCL5, GSK3β, THPO, GPC2, ANGPT2, PIGR, FST, HS6ST1, LGALS3, IL10, and LGMN. In some embodiments, a method comprises detecting the level of at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, at least seven biomarkers, or eight biomarkers selected from SIGLEC7, CCL5, IGFBP2, LTA LTB, PSPN, GSK3β, and IGFBP1. In some embodiments, a method comprises detecting the level of at least one biomarker, at least two biomarkers, at least three biomarkers, or four biomarkers selected from SIGLEC7, IGFBP1, PSPN, and GPC2. In some embodiments, a method comprises detecting the level of at least one biomarker, at least two biomarkers, or three biomarkers selected from SIGLEC7, CCL5, and IGFBP1. In some embodiments, a method comprises detecting SIGLEC7 and CCL5.

As used herein, “lung” may be interchangeably referred to as “pulmonary”.

As used herein, “smoker” refers to an individual who has a history of tobacco smoke inhalation.

As used herein, “pack year” refers to the number of packs of cigarettes per day that a smoker smokes times the number of years that the smoker has been smoking. For example, a former smoker that smoked two packs of cigarettes per day for ten years has a 20 pack year smoking history. As a further example, a current smoker who smokes one pack of cigarettes per day, and has done so for 20 years also has a 20 pack year smoking history.

“Biological sample”, “sample”, and “test sample” are used interchangeably herein to refer to any material, biological fluid, tissue, or cell obtained or otherwise derived from an individual. This includes blood (including whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, and serum), sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate (e.g., bronchoalveolar lavage), bronchial brushing, synovial fluid, joint aspirate, organ secretions, cells, a cellular extract, and cerebrospinal fluid. This also includes experimentally separated fractions of all of the preceding. For example, a blood sample can be fractionated into serum, plasma, or into fractions containing particular types of blood cells, such as red blood cells or white blood cells (leukocytes). In some embodiments, a sample can be a combination of samples from an individual, such as a combination of a tissue and fluid sample. The term “biological sample” also includes materials containing homogenized solid material, such as from a stool sample, a tissue sample, or a tissue biopsy, for example. The term “biological sample” also includes materials derived from a tissue culture or a cell culture. Any suitable methods for obtaining a biological sample can be employed; exemplary methods include, e.g., phlebotomy, swab (e.g., buccal swab), and a fine needle aspirate biopsy procedure. Exemplary tissues susceptible to fine needle aspiration include lymph node, lung, lung washes, BAL (bronchoalveolar lavage), thyroid, breast, pancreas, and liver. Samples can also be collected, e.g., by micro dissection (e.g., laser capture micro dissection (LCM) or laser micro dissection (LMD)), bladder wash, smear (e.g., a PAP smear), or ductal lavage. A “biological sample” obtained or derived from an individual includes any such sample that has been processed in any suitable manner after being obtained from the individual.

Further, in some embodiments, a biological sample may be derived by taking biological samples from a number of individuals and pooling them, or pooling an aliquot of each individual's biological sample. The pooled sample may be treated as described herein for a sample from a single individual, and, for example, if a poor prognosis is established in the pooled sample, then each individual biological sample can be re-tested to determine which individual(s) have COPD that is likely to progress and/or lung function that is predicted to decline.

“Target”, “target molecule”, and “analyte” are used interchangeably herein to refer to any molecule of interest that may be present in a biological sample. A “molecule of interest” includes any minor variation of a particular molecule, such as, in the case of a protein, for example, minor variations in amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component, which does not substantially alter the identity of the molecule. A “target molecule”, “target”, or “analyte” refers to a set of copies of one type or species of molecule or multi-molecular structure. “Target molecules”, “targets”, and “analytes” refer to more than one type or species of molecule or multi-molecular structure. Exemplary target molecules include proteins, polypeptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, affybodies, antibody mimics, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, and any fragment or portion of any of the foregoing. In some embodiments, a target molecule is a protein, in which case the target molecule may be referred to as a “target protein.”

As used herein, a “capture agent’ or “capture reagent” refers to a molecule that is capable of binding specifically to a biomarker. A “target protein capture reagent” refers to a molecule that is capable of binding specifically to a target protein. Nonlimiting exemplary capture reagents include aptamers, antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, autoantibodies, chimeras, small molecules, nucleic acids, lectins, ligand-binding receptors, imprinted polymers, avimers, peptidomimetics, hormone receptors, cytokine receptors, synthetic receptors, and modifications and fragments of any of the aforementioned capture reagents. In some embodiments, a capture reagent is selected from an aptamer and an antibody.

The term “antibody” refers to full-length antibodies of any species and fragments and derivatives of such antibodies, including Fab fragments, F(ab′)₂ fragments, single chain antibodies, Fv fragments, and single chain Fv fragments. The term “antibody” also refers to synthetically-derived antibodies, such as phage display-derived antibodies and fragments, affybodies, nanobodies, etc.

As used herein, “marker” and “biomarker” are used interchangeably to refer to a target molecule that indicates or is a sign of a normal or abnormal process in an individual or of a disease or other condition in an individual. More specifically, a “marker” or “biomarker” is an anatomic, physiologic, biochemical, or molecular parameter associated with the presence of a specific physiological state or process, whether normal or abnormal, and, if abnormal, whether chronic or acute. Biomarkers are detectable and measurable by a variety of methods including laboratory assays and medical imaging. In some embodiments, a biomarker is a target protein.

As used herein, “biomarker level” and “level” refer to a measurement that is made using any analytical method for detecting the biomarker in a biological sample and that indicates the presence, absence, absolute amount or concentration, relative amount or concentration, titer, a level, an expression level, a ratio of measured levels, or the like, of, for, or corresponding to the biomarker in the biological sample. The exact nature of the “level” depends on the specific design and components of the particular analytical method employed to detect the biomarker.

A “control level” of a target molecule refers to the level of the target molecule in the same sample type from an individual that does not have the disease or condition, or from an individual that is not suspected or at risk of having the disease or condition, or from an individual that has a non-progressive form of the disease or condition. A “control level” of a target molecule need not be determined each time the present methods are carried out, and may be a previously determined level that is used as a reference or threshold to determine whether the level in a particular sample is higher or lower than a normal level. In some embodiments, a control level in a method described herein is the level that has been observed in one or more subjects with non-progressive COPD. In some embodiments, a control level in a method described herein is the average or mean level, optionally plus or minus a statistical variation, that has been observed in a plurality of subjects with non-progressive COPD.

As used herein, “individual” and “subject” are used interchangeably to refer to a test subject or patient. The individual can be a mammal or a non-mammal. In various embodiments, the individual is a mammal. A mammalian individual can be a human or non-human. In various embodiments, the individual is a human. A healthy or normal individual is an individual in which the disease or condition of interest (such as COPD) is not detectable by conventional diagnostic methods.

“Diagnose”, “diagnosing”, “diagnosis”, and variations thereof refer to the detection, determination, or recognition of a health status or condition of an individual on the basis of one or more signs, symptoms, data, or other information pertaining to that individual. The health status of an individual can be diagnosed as healthy/normal (i.e., a diagnosis of the absence of a disease or condition) or diagnosed as ill/abnormal (i.e., a diagnosis of the presence, or an assessment of the characteristics, of a disease or condition). The terms “diagnose”, “diagnosing”, “diagnosis”, etc., encompass, with respect to a particular disease or condition, the initial detection of the disease; the characterization or classification of the disease; the detection of the progression, remission, or recurrence of the disease; and the detection of disease response after the administration of a treatment or therapy to the individual. The diagnosis of COPD includes distinguishing individuals, including smokers and nonsmokers, who have COPD from individuals who do not.

“Prognose”, “prognosing”, “prognosis”, and variations thereof refer to the prediction of a future course of a disease or condition in an individual who has the disease or condition (e.g., predicting patient survival), and such terms encompass the evaluation of disease response after the administration of a treatment or therapy to the individual.

“Evaluate”, “evaluating”, “evaluation”, and variations thereof encompass both “diagnose” and “prognose” and also encompass determinations or predictions about the future course of a disease or condition in an individual who does not have the disease as well as determinations or predictions regarding the likelihood that a disease or condition will recur in an individual who apparently has been cured of the disease. The term “evaluate” also encompasses assessing an individual's response to a therapy, such as, for example, predicting whether an individual is likely to respond favorably to a therapeutic agent or is unlikely to respond to a therapeutic agent (or will experience toxic or other undesirable side effects, for example), selecting a therapeutic agent for administration to an individual, or monitoring or determining an individual's response to a therapy that has been administered to the individual. Thus, “evaluating” COPD can include, for example, any of the following: prognosing the future course of COPD in an individual; predicting whether lung function in an individual with COPD will decline; predicting whether COPD in an individual will progress; determining whether a COPD treatment being administered is effective in the individual; or determining or predicting an individual's response to a COPD treatment or selecting a COPD treatment to administer to an individual based upon a determination of the biomarker levels derived from the individual's biological sample.

As used herein, “detecting” or “determining” with respect to a biomarker level includes the use of both the instrument used to observe and record a signal corresponding to a biomarker level and the material/s required to generate that signal. In various embodiments, the level is detected using any suitable method, including fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic waves, mass spectrometry, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, scanning tunneling microscopy, electrochemical detection methods, nuclear magnetic resonance, quantum dots, and the like.

As used herein, a “subject with chronic obstructive pulmonary disease (COPD)” refers to a subject that has been diagnosed with COPD. In some embodiments, COPD is diagnosed using the GOLD (global initiative for chronic obstructive lung disease) standard. See, e.g., Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: Updated 2007. Global Initiative for Chronic Obstructive Lung Disease (GOLD); available at www.goldcopd.org.

As used herein, a “subject at risk of developing COPD” refers to a subject with symptoms of COPD, such as chronic cough, chronic sputum production, and/or dyspnea; and/or a history of inhalation exposure to tobacco smoke or occupational dust.

“Progressive COPD”, “progressor COPD”, “COPD progressor” and the like are used interchangeably to refer to COPD in which lung function, as measured by FEV1%, decreases over time at a rate of at least 0.005 FEV1% per day. In other words, a plot of FEV1% versus time has a slope of ≦−0.005 FEV1%/day. COPD in which the plot of FEV1% versus time has a slope of >−0.005 FEV1%/day is considered to be “non-progressor COPD” or “non-progressive COPD”, etc. Similarly, lung function is considered to decline when FEV1% decreases at a rate of at least 0.005 FEV1% per day. Percent forced expiratory volume (FEV1%; also referred to as “FEV1% predicted”) is defined as the maximal amount of air forcefully exhaled in one second as compared to that of population norms for a person of the same height, weight, and gender. A normal FEV1% is greater than about 75%. FEV1 is typically measured by spirometry.

“Solid support” refers herein to any substrate having a surface to which molecules may be attached, directly or indirectly, through either covalent or non-covalent bonds. A “solid support” can have a variety of physical formats, which can include, for example, a membrane; a chip (e.g., a protein chip); a slide (e.g., a glass slide or coverslip); a column; a hollow, solid, semi-solid, pore- or cavity-containing particle, such as, for example, a bead; a gel; a fiber, including a fiber optic material; a matrix; and a sample receptacle. Exemplary sample receptacles include sample wells, tubes, capillaries, vials, and any other vessel, groove or indentation capable of holding a sample. A sample receptacle can be contained on a multi-sample platform, such as a microtiter plate, slide, microfluidics device, and the like. A support can be composed of a natural or synthetic material, an organic or inorganic material. The composition of the solid support on which capture reagents are attached generally depends on the method of attachment (e.g., covalent attachment). Other exemplary receptacles include microdroplets and microfluidic controlled or bulk oil/aqueous emulsions within which assays and related manipulations can occur. Suitable solid supports include, for example, plastics, resins, polysaccharides, silica or silica-based materials, functionalized glass, modified silicon, carbon, metals, inorganic glasses, membranes, nylon, natural fibers (such as, for example, silk, wool and cotton), polymers, and the like. The material composing the solid support can include reactive groups such as, for example, carboxy, amino, or hydroxyl groups, which are used for attachment of the capture reagents. Polymeric solid supports can include, e.g., polystyrene, polyethylene glycol tetraphthalate, polyvinyl acetate, polyvinyl chloride, polyvinyl pyrrolidone, polyacrylonitrile, polymethyl methacrylate, polytetrafluoroethylene, butyl rubber, styrenebutadiene rubber, natural rubber, polyethylene, polypropylene, (poly)tetrafluoroethylene, (poly)vinylidenefluoride, polycarbonate, and polymethylpentene. Suitable solid support particles that can be used include, e.g., encoded particles, such as Luminex-type encoded particles, magnetic particles, and glass particles.

Exemplary Uses of Biomarkers

In various exemplary embodiments, methods are provided for determining whether a subject with COPD has progressive COPD and/or determining whether a subject at risk of developing COPD will develop progressive COPD and/or determining whether lung function will decline in a subject with COPD or at risk of developing COPD. The methods comprise detecting one or more biomarker levels corresponding to one or more biomarkers that are present in the circulation of an individual, such as in serum or plasma, by any number of analytical methods, including any of the analytical methods described herein. These biomarkers are, for example, present at different levels in individuals with progressive COPD as compared to individuals with non-progressive COPD. Detection of the differential levels of a biomarker in an individual can be used, for example, to permit the determination of whether the individual has progressive COPD, will develop progressive COPD, and/or that lung function will decline in the individual. In some embodiments, any of the biomarkers described herein may be used to monitor COPD in an individual over time, and to permit the determination of whether the COPD is, or is likely to, progress. In some embodiments, any of the biomarkers described herein may be used to monitor an individual at risk of developing COPD over time, and to permit the determination of whether the individual is predicted to develop a progressive form of COPD.

As an example of the manner in which any of the biomarkers described herein can be used to predict whether an individual will develop a progressive form of COPD, levels of one or more of the described biomarkers in an individual who has not been diagnosed with COPD, but has one or more risk factors for COPD may indicate that the individual is likely to develop progressive COPD, thereby enabling detection of COPD at an early stage of the disease when treatment is more effective. In the case of biomarkers whose levels are higher in progressive COPD, in some embodiments, an increase in the level of one or more of the biomarkers during the course of follow-up treatment may be indicative of COPD progression, whereas a decrease in the level may indicate that the individual's COPD is moving toward or approaching a non-progressive form of the disease. Similarly, in some embodiments, for biomarkers whose levels are lower in progressive COPD, in some embodiments, a decrease in the level of one or more of the biomarkers during the course of follow-up treatment may be indicative of COPD progression, whereas an increase in the level may indicate that the individual's COPD is moving toward or approaching a non-progressive form of the disease. Furthermore, a differential expression level of one or more of the biomarkers in an individual over time may be indicative of the individual's response to a particular therapeutic regimen. In some embodiments, changes in expression of one or more of the biomarkers during follow-up monitoring may indicate that a particular therapy is effective or may suggest that the therapeutic regimen should be altered in some way, such as by changing one or more therapeutic agents and/or dosages.

In addition to testing biomarker levels as a stand-alone diagnostic test, biomarker levels can also be done in conjunction with determination of single nucleotide polymorphisms (SNPs) or other genetic lesions or variability that are indicative of increased risk of susceptibility of disease. (See, e.g., Amos et al., Nature Genetics 40, 616-622 (2009)).

In addition to testing biomarker levels as a stand-alone diagnostic test, biomarker levels can also be done in conjunction with other COPD screening methods, such as spirometry and imaging. For example, the biomarkers may facilitate the medical and economic justification for implementing more aggressive treatments for COPD, more frequent follow-up screening, etc. The biomarkers may also be used to begin treatment in individuals at risk of COPD, but who have not been diagnosed with COPD, if the diagnostic test indicates they are likely to develop a progressive form of the disease.

In addition to testing biomarker levels in conjunction with other COPD diagnostic methods, information regarding the biomarkers can also be evaluated in conjunction with other types of data, particularly data that indicates an individual's risk for COPD. These various data can be assessed by automated methods, such as a computer program/software, which can be embodied in a computer or other apparatus/device.

Detection and Determination of Biomarkers and Biomarker Levels

A biomarker level for the biomarkers described herein can be detected using any of a variety of known analytical methods. In one embodiment, a biomarker level is detected using a capture reagent. In various embodiments, the capture reagent can be exposed to the biomarker in solution or can be exposed to the biomarker while the capture reagent is immobilized on a solid support. In other embodiments, the capture reagent contains a feature that is reactive with a secondary feature on a solid support. In these embodiments, the capture reagent can be exposed to the biomarker in solution, and then the feature on the capture reagent can be used in conjunction with the secondary feature on the solid support to immobilize the biomarker on the solid support. The capture reagent is selected based on the type of analysis to be conducted. Capture reagents include but are not limited to aptamers, antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, autoantibodies, chimeras, small molecules, F(ab′)₂ fragments, single chain antibody fragments, Fv fragments, single chain Fv fragments, nucleic acids, lectins, ligand-binding receptors, affybodies, nanobodies, imprinted polymers, avimers, peptidomimetics, hormone receptors, cytokine receptors, and synthetic receptors, and modifications and fragments of these.

In some embodiments, a biomarker level is detected using a biomarker/capture reagent complex.

In some embodiments, the biomarker level is derived from the biomarker/capture reagent complex and is detected indirectly, such as, for example, as a result of a reaction that is subsequent to the biomarker/capture reagent interaction, but is dependent on the formation of the biomarker/capture reagent complex.

In some embodiments, the biomarker level is detected directly from the biomarker in a biological sample.

In some embodiments, biomarkers are detected using a multiplexed format that allows for the simultaneous detection of two or more biomarkers in a biological sample. In some embodiments of the multiplexed format, capture reagents are immobilized, directly or indirectly, covalently or non-covalently, in discrete locations on a solid support. In some embodiments, a multiplexed format uses discrete solid supports where each solid support has a unique capture reagent associated with that solid support, such as, for example quantum dots. In some embodiments, an individual device is used for the detection of each one of multiple biomarkers to be detected in a biological sample. Individual devices can be configured to permit each biomarker in the biological sample to be processed simultaneously. For example, a microtiter plate can be used such that each well in the plate is used to analyze one or more of multiple biomarkers to be detected in a biological sample.

In one or more of the foregoing embodiments, a fluorescent tag can be used to label a component of the biomarker/capture reagent complex to enable the detection of the biomarker level. In various embodiments, the fluorescent label can be conjugated to a capture reagent specific to any of the biomarkers described herein using known techniques, and the fluorescent label can then be used to detect the corresponding biomarker level. Suitable fluorescent labels include rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, allophycocyanin, PBXL-3, Qdot 605, Lissamine, phycoerythrin, Texas Red, and other such compounds.

In some embodiments, the fluorescent label is a fluorescent dye molecule. In some embodiments, the fluorescent dye molecule includes at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance. In some embodiments, the dye molecule includes an AlexFluor molecule, such as, for example, AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680, or AlexaFluor 700. In some embodiments, the dye molecule includes a first type and a second type of dye molecule, such as, e.g., two different AlexaFluor molecules. In some embodiments, the dye molecule includes a first type and a second type of dye molecule, and the two dye molecules have different emission spectra.

Fluorescence can be measured with a variety of instrumentation compatible with a wide range of assay formats. For example, spectrofluorimeters have been designed to analyze microtiter plates, microscope slides, printed arrays, cuvettes, etc. See Principles of Fluorescence Spectroscopy, by J. R. Lakowicz, Springer Science+Business Media, Inc., 2004. See Bioluminescence & Chemiluminescence: Progress & Current Applications; Philip E. Stanley and Larry J. Kricka editors, World Scientific Publishing Company, January 2002.

In one or more embodiments, a chemiluminescence tag can optionally be used to label a component of the biomarker/capture complex to enable the detection of a biomarker level. Suitable chemiluminescent materials include any of oxalyl chloride, Rodamin 6G, Ru(bipy)₃ ²⁺, TMAE (tetrakis(dimethylamino)ethylene), Pyrogallol (1,2,3-trihydroxibenzene), Lucigenin, peroxyoxalates, Aryl oxalates, Acridinium esters, dioxetanes, and others.

In some embodiments, the detection method includes an enzyme/substrate combination that generates a detectable signal that corresponds to the biomarker level. Generally, the enzyme catalyzes a chemical alteration of the chromogenic substrate which can be measured using various techniques, including spectrophotometry, fluorescence, and chemiluminescence. Suitable enzymes include, for example, luciferases, luciferin, malate dehydrogenase, urease, horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, uricase, xanthine oxidase, lactoperoxidase, microperoxidase, and the like.

In some embodiments, the detection method can be a combination of fluorescence, chemiluminescence, radionuclide or enzyme/substrate combinations that generate a measurable signal. In some embodiments, multimodal signaling could have unique and advantageous characteristics in biomarker assay formats.

In some embodiments, the biomarker levels for the biomarkers described herein can be detected using any analytical methods including, singleplex aptamer assays, multiplexed aptamer assays, singleplex or multiplexed immunoassays, mRNA expression profiling, miRNA expression profiling, mass spectrometric analysis, histological/cytological methods, etc. as discussed below.

Determination of Biomarker Levels Using Aptamer-Based Assays

Assays directed to the detection and quantification of physiologically significant molecules in biological samples and other samples are important tools in scientific research and in the health care field. One class of such assays involves the use of a microarray that includes one or more aptamers immobilized on a solid support. The aptamers are each capable of binding to a target molecule in a highly specific manner and with very high affinity. See, e.g., U.S. Pat. No. 5,475,096 entitled “Nucleic Acid Ligands”; see also, e.g., U.S. Pat. Nos. 6,242,246, 6,458,543, and 6,503,715, each of which is entitled “Nucleic Acid Ligand Diagnostic Biochip”. Once the microarray is contacted with a sample, the aptamers bind to their respective target molecules present in the sample and thereby enable a determination of a biomarker level corresponding to a biomarker.

As used herein, an “aptamer” refers to a nucleic acid that has a specific binding affinity for a target molecule. It is recognized that affinity interactions are a matter of degree; however, in this context, the “specific binding affinity” of an aptamer for its target means that the aptamer binds to its target generally with a much higher degree of affinity than it binds to other components in a test sample. An “aptamer” is a set of copies of one type or species of nucleic acid molecule that has a particular nucleotide sequence. An aptamer can include any suitable number of nucleotides, including any number of chemically modified nucleotides. “Aptamers” refers to more than one such set of molecules. Different aptamers can have either the same or different numbers of nucleotides. Aptamers can be DNA or RNA or chemically modified nucleic acids and can be single stranded, double stranded, or contain double stranded regions, and can include higher ordered structures. An aptamer can also be a photoaptamer, where a photoreactive or chemically reactive functional group is included in the aptamer to allow it to be covalently linked to its corresponding target. Any of the aptamer methods disclosed herein can include the use of two or more aptamers that specifically bind the same target molecule. As further described below, an aptamer may include a tag. If an aptamer includes a tag, all copies of the aptamer need not have the same tag. Moreover, if different aptamers each include a tag, these different aptamers can have either the same tag or a different tag.

An aptamer can be identified using any known method, including the SELEX process. Once identified, an aptamer can be prepared or synthesized in accordance with any known method, including chemical synthetic methods and enzymatic synthetic methods.

The terms “SELEX” and “SELEX process” are used interchangeably herein to refer generally to a combination of (1) the selection of aptamers that interact with a target molecule in a desirable manner, for example binding with high affinity to a protein, with (2) the amplification of those selected nucleic acids. The SELEX process can be used to identify aptamers with high affinity to a specific target or biomarker.

SELEX generally includes preparing a candidate mixture of nucleic acids, binding of the candidate mixture to the desired target molecule to form an affinity complex, separating the affinity complexes from the unbound candidate nucleic acids, separating and isolating the nucleic acid from the affinity complex, purifying the nucleic acid, and identifying a specific aptamer sequence. The process may include multiple rounds to further refine the affinity of the selected aptamer. The process can include amplification steps at one or more points in the process. See, e.g., U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands”. The SELEX process can be used to generate an aptamer that covalently binds its target as well as an aptamer that non-covalently binds its target. See, e.g., U.S. Pat. No. 5,705,337 entitled “Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Chemi-SELEX.”

The SELEX process can be used to identify high-affinity aptamers containing modified nucleotides that confer improved characteristics on the aptamer, such as, for example, improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified aptamers containing modified nucleotides are described in U.S. Pat. No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides”, which describes oligonucleotides containing nucleotide derivatives chemically modified at the 5′- and 2′-positions of pyrimidines. U.S. Pat. No. 5,580,737, see supra, describes highly specific aptamers containing one or more nucleotides modified with 2′-amino (2′-NH2), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). See also, U.S. Patent Application Publication No. 2009/0098549, entitled “SELEX and PHOTOSELEX”, which describes nucleic acid libraries having expanded physical and chemical properties and their use in SELEX and photoSELEX.

SELEX can also be used to identify aptamers that have desirable off-rate characteristics. See U.S. Publication No. US 2009/0004667, entitled “Method for Generating Aptamers with Improved Off-Rates”, which describes improved SELEX methods for generating aptamers that can bind to target molecules. Methods for producing aptamers and photoaptamers having slower rates of dissociation from their respective target molecules are described. The methods involve contacting the candidate mixture with the target molecule, allowing the formation of nucleic acid-target complexes to occur, and performing a slow off-rate enrichment process wherein nucleic acid-target complexes with fast dissociation rates will dissociate and not reform, while complexes with slow dissociation rates will remain intact. Additionally, the methods include the use of modified nucleotides in the production of candidate nucleic acid mixtures to generate aptamers with improved off-rate performance. Nonlimiting exemplary modified nucleotides include, for example, the modified pyrimidines shown in FIG. 12. In some embodiments, an aptamer comprises at least one nucleotide with a modification, such as a base modification. In some embodiments, an aptamer comprises at least one nucleotide with a hydrophobic modification, such as a hydrophobic base modification, allowing for hydrophobic contacts with a target protein. Such hydrophobic contacts, in some embodiments, contribute to greater affinity and/or slower off-rate binding by the aptamer. Nonlimiting exemplary nucleotides with hydrophobic modifications are shown in FIG. 12. In some embodiments, an aptamer comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 nucleotides with hydrophobic modifications, where each hydrophobic modification may be the same or different from the others. In some embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 hydrophobic modifications in an aptamer may be independently selected from the hydrophobic modifications shown in FIG. 12.

In some embodiments, a slow off-rate aptamer (including an aptamers comprising at least one nucleotide with a hydrophobic modification) has an off-rate (t_(1/2)) of ≧30 minutes, ≧60 minutes, ≧90 minutes, ≧120 minutes, ≧150 minutes, ≧180 minutes, ≧210 minutes, or ≧240 minutes.

In some embodiments, an assay employs aptamers that include photoreactive functional groups that enable the aptamers to covalently bind or “photocrosslink” their target molecules. See, e.g., U.S. Pat. No. 6,544,776 entitled “Nucleic Acid Ligand Diagnostic Biochip”. These photoreactive aptamers are also referred to as photoaptamers. See, e.g., U.S. Pat. Nos. 5,763,177, 6,001,577, and 6,291,184, each of which is entitled “Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX”; see also, e.g., U.S. Pat. No. 6,458,539, entitled “Photoselection of Nucleic Acid Ligands”. After the microarray is contacted with the sample and the photoaptamers have had an opportunity to bind to their target molecules, the photoaptamers are photoactivated, and the solid support is washed to remove any non-specifically bound molecules. Harsh wash conditions may be used, since target molecules that are bound to the photoaptamers are generally not removed, due to the covalent bonds created by the photoactivated functional group(s) on the photoaptamers. In this manner, the assay enables the detection of a biomarker level corresponding to a biomarker in the test sample.

In some assay formats, the aptamers are immobilized on the solid support prior to being contacted with the sample. Under certain circumstances, however, immobilization of the aptamers prior to contact with the sample may not provide an optimal assay. For example, pre-immobilization of the aptamers may result in inefficient mixing of the aptamers with the target molecules on the surface of the solid support, perhaps leading to lengthy reaction times and, therefore, extended incubation periods to permit efficient binding of the aptamers to their target molecules. Further, when photoaptamers are employed in the assay and depending upon the material utilized as a solid support, the solid support may tend to scatter or absorb the light used to effect the formation of covalent bonds between the photoaptamers and their target molecules. Moreover, depending upon the method employed, detection of target molecules bound to their aptamers can be subject to imprecision, since the surface of the solid support may also be exposed to and affected by any labeling agents that are used. Finally, immobilization of the aptamers on the solid support generally involves an aptamer-preparation step (i.e., the immobilization) prior to exposure of the aptamers to the sample, and this preparation step may affect the activity or functionality of the aptamers.

Aptamer assays that permit an aptamer to capture its target in solution and then employ separation steps that are designed to remove specific components of the aptamer-target mixture prior to detection have also been described (see U.S. Publication No. 2009/0042206, entitled “Multiplexed Analyses of Test Samples”). The described aptamer assay methods enable the detection and quantification of a non-nucleic acid target (e.g., a protein target) in a test sample by detecting and quantifying a nucleic acid (i.e., an aptamer). The described methods create a nucleic acid surrogate (i.e, the aptamer) for detecting and quantifying a non-nucleic acid target, thus allowing the wide variety of nucleic acid technologies, including amplification, to be applied to a broader range of desired targets, including protein targets.

Aptamers can be constructed to facilitate the separation of the assay components from an aptamer biomarker complex (or photoaptamer biomarker covalent complex) and permit isolation of the aptamer for detection and/or quantification. In one embodiment, these constructs can include a cleavable or releasable element within the aptamer sequence. In other embodiments, additional functionality can be introduced into the aptamer, for example, a labeled or detectable component, a spacer component, or a specific binding tag or immobilization element. For example, the aptamer can include a tag connected to the aptamer via a cleavable moiety, a label, a spacer component separating the label, and the cleavable moiety. In one embodiment, a cleavable element is a photocleavable linker. The photocleavable linker can be attached to a biotin moiety and a spacer section, can include an NHS group for derivatization of amines, and can be used to introduce a biotin group to an aptamer, thereby allowing for the release of the aptamer later in an assay method.

Homogenous assays, done with all assay components in solution, do not require separation of sample and reagents prior to the detection of signal. These methods are rapid and easy to use. These methods generate signal based on a molecular capture or binding reagent that reacts with its specific target. In some embodiments of the methods described herein, the molecular capture reagents comprise an aptamer or an antibody or the like and the specific target may be a biomarker shown in Table 4.

In some embodiments, a method for signal generation takes advantage of anisotropy signal change due to the interaction of a fluorophore-labeled capture reagent with its specific biomarker target. When the labeled capture reacts with its target, the increased molecular weight causes the rotational motion of the fluorophore attached to the complex to become much slower changing the anisotropy value. By monitoring the anisotropy change, binding events may be used to quantitatively measure the biomarkers in solutions. Other methods include fluorescence polarization assays, molecular beacon methods, time resolved fluorescence quenching, chemiluminescence, fluorescence resonance energy transfer, and the like.

An exemplary solution-based aptamer assay that can be used to detect a biomarker level in a biological sample includes the following: (a) preparing a mixture by contacting the biological sample with an aptamer that includes a first tag and has a specific affinity for the biomarker, wherein an aptamer affinity complex is formed when the biomarker is present in the sample; (b) exposing the mixture to a first solid support including a first capture element, and allowing the first tag to associate with the first capture element; (c) removing any components of the mixture not associated with the first solid support; (d) attaching a second tag to the biomarker component of the aptamer affinity complex; (e) releasing the aptamer affinity complex from the first solid support; (f) exposing the released aptamer affinity complex to a second solid support that includes a second capture element and allowing the second tag to associate with the second capture element; (g) removing any non-complexed aptamer from the mixture by partitioning the non-complexed aptamer from the aptamer affinity complex; (h) eluting the aptamer from the solid support; and (i) detecting the biomarker by detecting the aptamer component of the aptamer affinity complex.

A nonlimiting exemplary method of detecting biomarkers in a biological sample using aptamers is described in Example 7. See also Kraemer et al., PLoS One 6(10): e26332.

Determination of Biomarker Levels Using Immunoassays

Immunoassay methods are based on the reaction of an antibody to its corresponding target or analyte and can detect the analyte in a sample depending on the specific assay format. To improve specificity and sensitivity of an assay method based on immuno-reactivity, monoclonal antibodies and fragments thereof are often used because of their specific epitope recognition. Polyclonal antibodies have also been successfully used in various immunoassays because of their increased affinity for the target as compared to monoclonal antibodies Immunoassays have been designed for use with a wide range of biological sample matrices Immunoassay formats have been designed to provide qualitative, semi-quantitative, and quantitative results.

Quantitative results are generated through the use of a standard curve created with known concentrations of the specific analyte to be detected. The response or signal from an unknown sample is plotted onto the standard curve, and a quantity or level corresponding to the target in the unknown sample is established.

Numerous immunoassay formats have been designed. ELISA or EIA can be quantitative for the detection of an analyte. This method relies on attachment of a label to either the analyte or the antibody and the label component includes, either directly or indirectly, an enzyme. ELISA tests may be formatted for direct, indirect, competitive, or sandwich detection of the analyte. Other methods rely on labels such as, for example, radioisotopes (I¹²⁵) or fluorescence. Additional techniques include, for example, agglutination, nephelometry, turbidimetry, Western blot, immunoprecipitation, immunocytochemistry, immunohistochemistry, flow cytometry, Luminex assay, and others (see ImmunoAssay: A Practical Guide, edited by Brian Law, published by Taylor & Francis, Ltd., 2005 edition).

Exemplary assay formats include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, fluorescent, chemiluminescence, and fluorescence resonance energy transfer (FRET) or time resolved-FRET (TR-FRET) immunoassays. Examples of procedures for detecting biomarkers include biomarker immunoprecipitation followed by quantitative methods that allow size and peptide level discrimination, such as gel electrophoresis, capillary electrophoresis, planar electrochromatography, and the like.

Methods of detecting and/or for quantifying a detectable label or signal generating material depend on the nature of the label. The products of reactions catalyzed by appropriate enzymes (where the detectable label is an enzyme; see above) can be, without limitation, fluorescent, luminescent, or radioactive or they may absorb visible or ultraviolet light. Examples of detectors suitable for detecting such detectable labels include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers.

Any of the methods for detection can be performed in any format that allows for any suitable preparation, processing, and analysis of the reactions. This can be, for example, in multi-well assay plates (e.g., 96 wells or 386 wells) or using any suitable array or microarray. Stock solutions for various agents can be made manually or robotically, and all subsequent pipetting, diluting, mixing, distribution, washing, incubating, sample readout, data collection and analysis can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting a detectable label.

Determination of Biomarker Levels Using Gene Expression Profiling

Measuring mRNA in a biological sample may, in some embodiments, be used as a surrogate for detection of the level of the corresponding protein in the biological sample. Thus, in some embodiments, a biomarker or biomarker panel described herein can be detected by detecting the appropriate RNA.

In some embodiments, mRNA expression levels are measured by reverse transcription quantitative polymerase chain reaction (RT-PCR followed with qPCR). RT-PCR is used to create a cDNA from the mRNA. The cDNA may be used in a qPCR assay to produce fluorescence as the DNA amplification process progresses. By comparison to a standard curve, qPCR can produce an absolute measurement such as number of copies of mRNA per cell. Northern blots, microarrays, Invader assays, and RT-PCR combined with capillary electrophoresis have all been used to measure expression levels of mRNA in a sample. See Gene Expression Profiling: Methods and Protocols, Richard A. Shimkets, editor, Humana Press, 2004.

Detection of Biomarkers Using In Vivo Molecular Imaging Technologies

In some embodiments, a biomarker described herein may be used in molecular imaging tests. For example, an imaging agent can be coupled to a capture reagent, which can be used to detect the biomarker in vivo.

In vivo imaging technologies provide non-invasive methods for determining the state of a particular disease in the body of an individual. For example, entire portions of the body, or even the entire body, may be viewed as a three dimensional image, thereby providing valuable information concerning morphology and structures in the body. Such technologies may be combined with the detection of the biomarkers described herein to provide information concerning the biomarker in vivo.

The use of in vivo molecular imaging technologies is expanding due to various advances in technology. These advances include the development of new contrast agents or labels, such as radiolabels and/or fluorescent labels, which can provide strong signals within the body; and the development of powerful new imaging technology, which can detect and analyze these signals from outside the body, with sufficient sensitivity and accuracy to provide useful information. The contrast agent can be visualized in an appropriate imaging system, thereby providing an image of the portion or portions of the body in which the contrast agent is located. The contrast agent may be bound to or associated with a capture reagent, such as an aptamer or an antibody, for example, and/or with a peptide or protein, or an oligonucleotide (for example, for the detection of gene expression), or a complex containing any of these with one or more macromolecules and/or other particulate forms.

The contrast agent may also feature a radioactive atom that is useful in imaging. Suitable radioactive atoms include technetium-99m or iodine-123 for scintigraphic studies. Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI) such as, for example, iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Such labels are well known in the art and could easily be selected by one of ordinary skill in the art.

Standard imaging techniques include but are not limited to magnetic resonance imaging, computed tomography scanning, positron emission tomography (PET), single photon emission computed tomography (SPECT), and the like. For diagnostic in vivo imaging, the type of detection instrument available is a major factor in selecting a given contrast agent, such as a given radionuclide and the particular biomarker that it is used to target (protein, mRNA, and the like). The radionuclide chosen typically has a type of decay that is detectable by a given type of instrument. Also, when selecting a radionuclide for in vivo diagnosis, its half-life should be long enough to enable detection at the time of maximum uptake by the target tissue but short enough that deleterious radiation of the host is minimized.

Exemplary imaging techniques include but are not limited to PET and SPECT, which are imaging techniques in which a radionuclide is synthetically or locally administered to an individual. The subsequent uptake of the radiotracer is measured over time and used to obtain information about the targeted tissue and the biomarker. Because of the high-energy (gamma-ray) emissions of the specific isotopes employed and the sensitivity and sophistication of the instruments used to detect them, the two-dimensional distribution of radioactivity may be inferred from outside of the body.

Commonly used positron-emitting nuclides in PET include, for example, carbon-11, nitrogen-13, oxygen-15, and fluorine-18. Isotopes that decay by electron capture and/or gamma-emission are used in SPECT and include, for example iodine-123 and technetium-99m. An exemplary method for labeling amino acids with technetium-99m is the reduction of pertechnetate ion in the presence of a chelating precursor to form the labile technetium-99m-precursor complex, which, in turn, reacts with the metal binding group of a bifunctionally modified chemotactic peptide to form a technetium-99m-chemotactic peptide conjugate.

Antibodies are frequently used for such in vivo imaging diagnostic methods. The preparation and use of antibodies for in vivo diagnosis is well known in the art. Similarly, aptamers may be used for such in vivo imaging diagnostic methods. For example, an aptamer that was used to identify a particular biomarker described herein may be appropriately labeled and injected into an individual to detect the biomarker in vivo. The label used will be selected in accordance with the imaging modality to be used, as previously described. Aptamer-directed imaging agents could have unique and advantageous characteristics relating to tissue penetration, tissue distribution, kinetics, elimination, potency, and selectivity as compared to other imaging agents.

Such techniques may also optionally be performed with labeled oligonucleotides, for example, for detection of gene expression through imaging with antisense oligonucleotides. These methods are used for in situ hybridization, for example, with fluorescent molecules or radionuclides as the label. Other methods for detection of gene expression include, for example, detection of the activity of a reporter gene.

Another general type of imaging technology is optical imaging, in which fluorescent signals within the subject are detected by an optical device that is external to the subject. These signals may be due to actual fluorescence and/or to bioluminescence. Improvements in the sensitivity of optical detection devices have increased the usefulness of optical imaging for in vivo diagnostic assays.

For a review of other techniques, see N. Blow, Nature Methods, 6, 465-469, 2009.

Determination of Biomarkers Using Histology/Cytology Methods

In some embodiments, the biomarkers described herein may be detected in a variety of tissue samples using histological or cytological methods. For example, endo- and trans-bronchial biopsies, fine needle aspirates, cutting needles, and core biopsies can be used for histology. Bronchial washing and brushing, pleural aspiration, and sputum, can be used for cyotology. Any of the biomarkers identified herein can be used to stain a specimen as an indication of disease.

In some embodiments, one or more capture reagent/s specific to the corresponding biomarker/s are used in a cytological evaluation of a sample and may include one or more of the following: collecting a cell sample, fixing the cell sample, dehydrating, clearing, immobilizing the cell sample on a microscope slide, permeabilizing the cell sample, treating for analyte retrieval, staining, destaining, washing, blocking, and reacting with one or more capture reagent/s in a buffered solution. In another embodiment, the cell sample is produced from a cell block.

In some embodiments, one or more capture reagent/s specific to the corresponding biomarkers are used in a histological evaluation of a tissue sample and may include one or more of the following: collecting a tissue specimen, fixing the tissue sample, dehydrating, clearing, immobilizing the tissue sample on a microscope slide, permeabilizing the tissue sample, treating for analyte retrieval, staining, destaining, washing, blocking, rehydrating, and reacting with capture reagent/s in a buffered solution. In another embodiment, fixing and dehydrating are replaced with freezing.

In another embodiment, the one or more aptamer/s specific to the corresponding biomarker/s are reacted with the histological or cytological sample and can serve as the nucleic acid target in a nucleic acid amplification method. Suitable nucleic acid amplification methods include, for example, PCR, q-beta replicase, rolling circle amplification, strand displacement, helicase dependent amplification, loop mediated isothermal amplification, ligase chain reaction, and restriction and circularization aided rolling circle amplification.

In one embodiment, the one or more capture reagent/s specific to the corresponding biomarkers for use in the histological or cytological evaluation are mixed in a buffered solution that can include any of the following: blocking materials, competitors, detergents, stabilizers, carrier nucleic acid, polyanionic materials, etc.

A “cytology protocol” generally includes sample collection, sample fixation, sample immobilization, and staining. “Cell preparation” can include several processing steps after sample collection, including the use of one or more aptamers for the staining of the prepared cells.

Determination of Biomarker Levels Using Mass Spectrometry Methods

A variety of configurations of mass spectrometers can be used to detect biomarker levels. Several types of mass spectrometers are available or can be produced with various configurations. In general, a mass spectrometer has the following major components: a sample inlet, an ion source, a mass analyzer, a detector, a vacuum system, and instrument-control system, and a data system. Difference in the sample inlet, ion source, and mass analyzer generally define the type of instrument and its capabilities. For example, an inlet can be a capillary-column liquid chromatography source or can be a direct probe or stage such as used in matrix-assisted laser desorption. Common ion sources are, for example, electrospray, including nanospray and microspray or matrix-assisted laser desorption. Common mass analyzers include a quadrupole mass filter, ion trap mass analyzer and time-of-flight mass analyzer. Additional mass spectrometry methods are well known in the art (see Burlingame et al. Anal. Chem. 70:647 R-716R (1998); Kinter and Sherman, New York (2000)).

Protein biomarkers and biomarker levels can be detected and measured by any of the following: electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), tandem time-of-flight (TOF/TOF) technology, called ultraflex III TOF/TOF, atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)^(N), atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)^(N), quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), quantitative mass spectrometry, and ion trap mass spectrometry.

Sample preparation strategies are used to label and enrich samples before mass spectroscopic characterization of protein biomarkers and determination biomarker levels. Labeling methods include but are not limited to isobaric tag for relative and absolute quantitation (iTRAQ) and stable isotope labeling with amino acids in cell culture (SILAC). Capture reagents used to selectively enrich samples for candidate biomarker proteins prior to mass spectroscopic analysis include but are not limited to aptamers, antibodies, nucleic acid probes, chimeras, small molecules, an F(ab′)₂ fragment, a single chain antibody fragment, an Fv fragment, a single chain Fv fragment, a nucleic acid, a lectin, a ligand-binding receptor, affybodies, nanobodies, ankyrins, domain antibodies, alternative antibody scaffolds (e.g. diabodies etc) imprinted polymers, avimers, peptidomimetics, peptoids, peptide nucleic acids, threose nucleic acid, a hormone receptor, a cytokine receptor, and synthetic receptors, and modifications and fragments of these.

The foregoing assays enable the detection of biomarker levels that are useful in the methods described herein, where the methods comprise detecting, in a biological sample from an individual, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine biomarkers selected from the biomarkers in Table 4 or Table 6. In various embodiments, the methods comprise detecting the levels of one or more biomarkers selected from any of the groups of biomarkers described herein, such as the panels shown in Tables 3, 7, and 8 and the subsets of biomarkers of Table 4 or Table 6 discussed throughout this application. Thus, while some of the described biomarkers may be useful alone for detecting progressive COPD, methods are also described herein for the grouping of multiple biomarkers and subsets of the biomarkers to form panels of two or more biomarkers. In accordance with any of the methods described herein, biomarker levels can be detected and classified individually or they can be detected and classified collectively, as for example in a multiplex assay format.

Classification of Biomarkers and Calculation of Disease Scores

In some embodiments, a biomarker “signature” for a given diagnostic test contains a set of markers, each marker having characteristic levels in the populations of interest. Characteristic levels, in some embodiments, may refer to the mean or average of the biomarker levels for the individuals in a particular group. In some embodiments, a diagnostic method described herein can be used to assign an unknown sample from an individual into one of two groups, either progressive COPD or non-progressive COPD; or alternatively, lung function likely to decline or lung function less likely to decline. The assignment of a sample into one of two or more groups is known as classification, and the procedure used to accomplish this assignment is known as a classifier or a classification method. Classification methods may also be referred to as scoring methods. There are many classification methods that can be used to construct a diagnostic classifier from a set of biomarker levels. In some instances, classification methods are performed using supervised learning techniques in which a data set is collected using samples obtained from individuals within two (or more, for multiple classification states) distinct groups one wishes to distinguish. Since the class (group or population) to which each sample belongs is known in advance for each sample, the classification method can be trained to give the desired classification response. It is also possible to use unsupervised learning techniques to produce a diagnostic classifier.

Common approaches for developing diagnostic classifiers include decision trees; bagging+boosting+forests; rule inference based learning; Parzen Windows; linear models; logistic; neural network methods; unsupervised clustering; K-means; hierarchical ascending/descending; semi-supervised learning; prototype methods; nearest neighbor; kernel density estimation; support vector machines; hidden Markov models; Boltzmann Learning; and classifiers may be combined either simply or in ways which minimize particular objective functions. For a review, see, e.g., Pattern Classification, R. O. Duda, et al., editors, John Wiley & Sons, 2nd edition, 2001; see also, The Elements of Statistical Learning—Data Mining, Inference, and Prediction, T. Hastie, et al., editors, Springer Science+Business Media, LLC, 2nd edition, 2009.

To produce a classifier using supervised learning techniques, a set of samples called training data are obtained. In the context of diagnostic tests, training data includes samples from the distinct groups (classes) to which unknown samples will later be assigned. For example, samples collected from individuals in a control population and individuals in a particular disease population can constitute training data to develop a classifier that can classify unknown samples (or, more particularly, the individuals from whom the samples were obtained) as either having the disease or being free from the disease. The development of the classifier from the training data is known as training the classifier. Specific details on classifier training depend on the nature of the supervised learning technique. Training a naïve Bayesian classifier is an example of such a supervised learning technique (see, e.g., Pattern Classification, R. O. Duda, et al., editors, John Wiley & Sons, 2nd edition, 2001; see also, The Elements of Statistical Learning—Data Mining, Inference, and Prediction, T. Hastie, et al., editors, Springer Science+Business Media, LLC, 2nd edition, 2009). Training of a naïve Bayesian classifier is described, e.g., in U.S. Publication Nos: 2012/0101002 and 2012/0077695.

Since typically there are many more potential biomarker levels than samples in a training set, care must be used to avoid over-fitting. Over-fitting occurs when a statistical model describes random error or noise instead of the underlying relationship. Over-fitting can be avoided in a variety of way, including, for example, by limiting the number of markers used in developing the classifier, by assuming that the marker responses are independent of one another, by limiting the complexity of the underlying statistical model employed, and by ensuring that the underlying statistical model conforms to the data.

An illustrative example of the development of a diagnostic test using a set of biomarkers includes the application of a naïve Bayes classifier, a simple probabilistic classifier based on Bayes theorem with strict independent treatment of the biomarkers. Each biomarker is described by a class-dependent probability density function (pdf) for the measured RFU values or log RFU (relative fluorescence units) values in each class. The joint pdfs for the set of markers in one class is assumed to be the product of the individual class-dependent pdfs for each biomarker. Training a naïve Bayes classifier in this context amounts to assigning parameters (“parameterization”) to characterize the class dependent pdfs. Any underlying model for the class-dependent pdfs may be used, but the model should generally conform to the data observed in the training set.

The performance of the naïve Bayes classifier is dependent upon the number and quality of the biomarkers used to construct and train the classifier. A single biomarker will perform in accordance with its KS-distance (Kolmogorov-Smirnov). The addition of subsequent markers with good KS distances (>0.3, for example) will, in general, improve the classification performance if the subsequently added markers are independent of the first marker. Using the sensitivity plus specificity as a classifier score, many high scoring classifiers can be generated with a variation of a greedy algorithm. (A greedy algorithm is any algorithm that follows the problem solving metaheuristic of making the locally optimal choice at each stage with the hope of finding the global optimum.)

Another way to depict classifier performance is through a receiver operating characteristic (ROC), or simply ROC curve or ROC plot. The ROC is a graphical plot of the sensitivity, or true positive rate, vs. false positive rate (1—specificity or 1—true negative rate), for a binary classifier system as its discrimination threshold is varied. The ROC can also be represented equivalently by plotting the fraction of true positives out of the positives (TPR=true positive rate) vs. the fraction of false positives out of the negatives (FPR=false positive rate). Also known as a Relative Operating Characteristic curve, because it is a comparison of two operating characteristics (TPR & FPR) as the criterion changes. The area under the ROC curve (AUC) is commonly used as a summary measure of diagnostic accuracy. It can take values from 0.0 to 1.0. The AUC has an important statistical property: the AUC of a classifier is equivalent to the probability that the classifier will rank a randomly chosen positive instance higher than a randomly chosen negative instance (Fawcett T, 2006. An introduction to ROC analysis. Pattern Recognition Letters .27: 861-874). This is equivalent to the Wilcoxon test of ranks (Hanley, J. A., McNeil, B. J., 1982. The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology 143, 29-36.).

Exemplary embodiments use any number of the biomarkers listed in Table 4 in various combinations to produce diagnostic tests for identifying individuals with progressive COPD. The markers listed in Table 4 can be combined in many ways to produce classifiers. Similarly, exemplary embodiments use any number of the biomarkers listed in Table 6 in various combinations to produce diagnostic tests for identifying individuals with progressive COPD. The markers listed in Table 6 can be combined in many ways to produce classifiers. In some embodiments, panels of biomarkers are comprised of different sets of biomarkers depending on a specific diagnostic performance criterion that is selected. For example, certain combinations of biomarkers may produce tests that are more sensitive (or more specific) than other combinations.

In some embodiments, once a panel is defined to include a particular set of biomarkers from Table 4 or Table 6 and a classifier is constructed from a set of training data, the diagnostic test parameters are complete. In some embodiments, a biological sample is run in one or more assays to produce the relevant quantitative biomarker levels used for classification. The measured biomarker levels are used as input for the classification method that outputs a classification and an optional score for the sample that reflects the confidence of the class assignment.

In some embodiments, a biological sample is optionally diluted and run in a multiplexed aptamer assay, and data is assessed as follows. First, the data from the assay are optionally normalized and calibrated, and the resulting biomarker levels are used as input to a Bayes classification scheme. Second, the log-likelihood ratio is computed for each measured biomarker individually and then summed to produce a final classification score, which is also referred to as a diagnostic score. The resulting assignment as well as the overall classification score can be reported. In some embodiments, the individual log-likelihood risk factors computed for each biomarker level can be reported as well.

Kits

Any combination of the biomarkers described herein can be detected using a suitable kit, such as for use in performing the methods disclosed herein. Furthermore, any kit can contain one or more detectable labels as described herein, such as a fluorescent moiety, etc.

In some embodiments, a kit includes (a) one or more capture reagents (such as, for example, at least one aptamer or antibody) for detecting one or more biomarkers in a biological sample, and optionally (b) one or more software or computer program products for predicting whether the individual from whom the biological sample was obtained has progressive COPD and/or whether lung function will decline in the individual. Alternatively, rather than one or more computer program products, one or more instructions for manually performing the above steps by a human can be provided.

In some embodiments, a kit comprises a solid support, a capture reagent, and a signal generating material. The kit can also include instructions for using the devices and reagents, handling the sample, and analyzing the data. Further the kit may be used with a computer system or software to analyze and report the result of the analysis of the biological sample.

The kits can also contain one or more reagents (e.g., solubilization buffers, detergents, washes, or buffers) for processing a biological sample. Any of the kits described herein can also include, e.g., buffers, blocking agents, mass spectrometry matrix materials, antibody capture agents, positive control samples, negative control samples, software and information such as protocols, guidance and reference data.

In some embodiments, kits are provided for the analysis of COPD, wherein the kits comprise PCR primers for one or more biomarkers described herein. In some embodiments, a kit may further include instructions for use and correlation of the biomarkers with COPD prognosis. In some embodiments, a kit may include a DNA array containing the complement of one or more of the biomarkers described herein, reagents, and/or enzymes for amplifying or isolating sample DNA. The kits may include reagents for real-time PCR, for example, TaqMan probes and/or primers, and enzymes.

For example, a kit can comprise (a) reagents comprising at least one capture reagent for determining the level of one or more biomarkers in a test sample, and optionally (b) one or more algorithms or computer programs for performing the steps of comparing the amount of each biomarker quantified in the test sample to one or more predetermined cutoffs. In some embodiments, an algorithm or computer program assigns a score for each biomarker quantified based on said comparison and, in some embodiments, combines the assigned scores for each biomarker quantified to obtain a total score. Further, in some embodiments, an algorithm or computer program compares the total score with a predetermined score, and uses the comparison to determine whether the COPD is likely to progress and/or whether lung function in the subject is likely to decline. Alternatively, rather than one or more algorithms or computer programs, one or more instructions for manually performing the above steps by a human can be provided.

Computer Methods and Software

Once a biomarker or biomarker panel is selected, a method for assessing COPD in an individual may comprise the following: 1) collect or otherwise obtain a biological sample; 2) perform an analytical method to detect and measure the biomarker or biomarkers in the panel in the biological sample; and 3) report the results of the biomarker levels. In some embodiments, the results of the biomarker levels are reported qualitatively rather than quantitatively, such as, for example, a proposed diagnosis (“progressive COPD”, “lung function likely to decline,” etc.) or simply a positive/negative result where “positive” and “negative” are defined. In some embodiments, a method for assessing COPD in an individual may comprise the following: 1) collect or otherwise obtain a biological sample; 2) perform an analytical method to detect and measure the biomarker or biomarkers in the panel in the biological sample; 3) perform any data normalization or standardization; 4) calculate each biomarker level; and 5) report the results of the biomarker levels. In some embodiments, the biomarker levels are combined in some way and a single value for the combined biomarker levels is reported. In this approach, in some embodiments, the reported value may be a single number determined from the sum of all the marker calculations that is compared to a pre-set threshold value that is an indication of the presence or absence of disease. Or the diagnostic score may be a series of bars that each represent a biomarker value and the pattern of the responses may be compared to a pre-set pattern for determination of the presence or absence of disease.

At least some embodiments of the methods described herein can be implemented with the use of a computer. An example of a computer system 100 is shown in FIG. 10. With reference to FIG. 10, system 100 is shown comprised of hardware elements that are electrically coupled via bus 108, including a processor 101, input device 102, output device 103, storage device 104, computer-readable storage media reader 105 a, communications system 106 processing acceleration (e.g., DSP or special-purpose processors) 107 and memory 109. Computer-readable storage media reader 105 a is further coupled to computer-readable storage media 105 b, the combination comprehensively representing remote, local, fixed and/or removable storage devices plus storage media, memory, etc. for temporarily and/or more permanently containing computer-readable information, which can include storage device 104, memory 109 and/or any other such accessible system 100 resource. System 100 also comprises software elements (shown as being currently located within working memory 191) including an operating system 192 and other code 193, such as programs, data and the like.

With respect to FIG. 10, system 100 has extensive flexibility and configurability. Thus, for example, a single architecture might be utilized to implement one or more servers that can be further configured in accordance with currently desirable protocols, protocol variations, extensions, etc. However, it will be apparent to those skilled in the art that embodiments may well be utilized in accordance with more specific application requirements. For example, one or more system elements might be implemented as sub-elements within a system 100 component (e.g., within communications system 106). Customized hardware might also be utilized and/or particular elements might be implemented in hardware, software or both. Further, while connection to other computing devices such as network input/output devices (not shown) may be employed, it is to be understood that wired, wireless, modem, and/or other connection or connections to other computing devices might also be utilized.

In one aspect, the system can comprise a database containing features of biomarkers characteristic of progressive COPD. The biomarker data (or biomarker information) can be utilized as an input to the computer for use as part of a computer implemented method. The biomarker data can include the data as described herein.

In one aspect, the system further comprises one or more devices for providing input data to the one or more processors.

The system further comprises a memory for storing a data set of ranked data elements.

In another aspect, the device for providing input data comprises a detector for detecting the characteristic of the data element, e.g., such as a mass spectrometer or gene chip reader.

The system additionally may comprise a database management system. User requests or queries can be formatted in an appropriate language understood by the database management system that processes the query to extract the relevant information from the database of training sets.

The system may be connectable to a network to which a network server and one or more clients are connected. The network may be a local area network (LAN) or a wide area network (WAN), as is known in the art. Preferably, the server includes the hardware necessary for running computer program products (e.g., software) to access database data for processing user requests.

The system may include an operating system (e.g., UNIX® or Linux) for executing instructions from a database management system. In one aspect, the operating system can operate on a global communications network, such as the internet, and utilize a global communications network server to connect to such a network.

The system may include one or more devices that comprise a graphical display interface comprising interface elements such as buttons, pull down menus, scroll bars, fields for entering text, and the like as are routinely found in graphical user interfaces known in the art. Requests entered on a user interface can be transmitted to an application program in the system for formatting to search for relevant information in one or more of the system databases. Requests or queries entered by a user may be constructed in any suitable database language.

The graphical user interface may be generated by a graphical user interface code as part of the operating system and can be used to input data and/or to display inputted data. The result of processed data can be displayed in the interface, printed on a printer in communication with the system, saved in a memory device, and/or transmitted over the network or can be provided in the form of the computer readable medium.

The system can be in communication with an input device for providing data regarding data elements to the system (e.g., expression values). In one aspect, the input device can include a gene expression profiling system including, e.g., a mass spectrometer, gene chip or array reader, and the like.

The methods and apparatus for analyzing biomarker information according to various embodiments may be implemented in any suitable manner, for example, using a computer program operating on a computer system. A conventional computer system comprising a processor and a random access memory, such as a remotely-accessible application server, network server, personal computer or workstation may be used. Additional computer system components may include memory devices or information storage systems, such as a mass storage system and a user interface, for example a conventional monitor, keyboard and tracking device. The computer system may be a stand-alone system or part of a network of computers including a server and one or more databases.

The biomarker analysis system can provide functions and operations to complete data analysis, such as data gathering, processing, analysis, reporting and/or diagnosis. For example, in one embodiment, the computer system can execute the computer program that may receive, store, search, analyze, and report information relating to the biomarkers. The computer program may comprise multiple modules performing various functions or operations, such as a processing module for processing raw data and generating supplemental data and an analysis module for analyzing raw data and supplemental data to generate a disease status and/or diagnosis. Identifying progressive COPD may comprise generating or collecting any other information, including additional biomedical information, regarding the condition of the individual relative to the disease, identifying whether further tests may be desirable, or otherwise evaluating the health status of the individual.

Some embodiments described herein can be implemented so as to include a computer program product. A computer program product may include a computer readable medium having computer readable program code embodied in the medium for causing an application program to execute on a computer with a database.

As used herein, a “computer program product” refers to an organized set of instructions in the form of natural or programming language statements that are contained on a physical media of any nature (e.g., written, electronic, magnetic, optical or otherwise) and that may be used with a computer or other automated data processing system. Such programming language statements, when executed by a computer or data processing system, cause the computer or data processing system to act in accordance with the particular content of the statements. Computer program products include without limitation: programs in source and object code and/or test or data libraries embedded in a computer readable medium. Furthermore, the computer program product that enables a computer system or data processing equipment device to act in pre-selected ways may be provided in a number of forms, including, but not limited to, original source code, assembly code, object code, machine language, encrypted or compressed versions of the foregoing and any and all equivalents.

In one aspect, a computer program product is provided for indicating whether COPD is likely to progress and/or whether lung function is likely to decline. The computer program product includes a computer readable medium embodying program code executable by a processor of a computing device or system, the program code comprising: code that retrieves data attributed to a biological sample from an individual, wherein the data comprises biomarker levels that correspond to one or more of the biomarkers described herein, and code that executes a classification method that indicates the COPD status of the individual as a function of the biomarker levels.

While various embodiments have been described as methods or apparatuses, it should be understood that embodiments can be implemented through code coupled with a computer, e.g., code resident on a computer or accessible by the computer. For example, software and databases could be utilized to implement many of the methods discussed above. Thus, in addition to embodiments accomplished by hardware, it is also noted that these embodiments can be accomplished through the use of an article of manufacture comprised of a computer usable medium having a computer readable program code embodied therein, which causes the enablement of the functions disclosed in this description. Therefore, it is desired that embodiments also be considered protected by this patent in their program code means as well. Furthermore, the embodiments may be embodied as code stored in a computer-readable memory of virtually any kind including, without limitation, RAM, ROM, magnetic media, optical media, or magneto-optical media. Even more generally, the embodiments could be implemented in software, or in hardware, or any combination thereof including, but not limited to, software running on a general purpose processor, microcode, programmable logic arrays (PLAs), or application-specific integrated circuits (ASICs).

It is also envisioned that embodiments could be accomplished as computer signals embodied in a carrier wave, as well as signals (e.g., electrical and optical) propagated through a transmission medium. Thus, the various types of information discussed above could be formatted in a structure, such as a data structure, and transmitted as an electrical signal through a transmission medium or stored on a computer readable medium.

Methods of Treatment

In some embodiments, following a determination that lung function is likely to decline in a subject with COPD, the subject is treated to reduce the rate of lung function decline. In some embodiments, following a determination that lung function is likely to decline in a subject at risk of developing COPD, the subject is treated to reduce the rate of lung function decline. In some embodiments, following a determination that COPD in a subject will progress, the subject is treated to slow the progression of COPD. In some embodiments, following a determination that a subject is at risk for developing progressive COPD, a subject is treated to slow the development of progressive COPD.

Treatments to reduce the rate of lung function decline, slow the progression of COPD, and/or slow the development of progressive COPD include, but are not limited to, treatments for COPD. Nonlimiting exemplary treatments for COPD include smoking cessation programs, long-acting bronchodilators, inhaled corticosteroids, and systemic corticosteroids. Smoking cessation programs may include therapeutic agents that aid in smoking cessation (including, but not limited to, bupropion, clonidine, topiramate, tryptophan, varenicline, notriptyline, and nicotine) and behavior modification therapy (including, but not limited to, hypnosis, behavior modification based on the Stages of Change model (see, e.g., Zimmerman et al., Am Fam Physician. 2000 Mar. 1; 61(5):1409-1416), and so-called 12-step programs, such as Nicotine Anonymous). Exemplary long-acting bronchodilators include, but are not limited to, β2-agonists (including, but not limited to, salmeterol, formoterol, bambuterol, and indacaterol) and anticholinergics (including, but not limited to, tiotropium and ipratropium bromide), and theophylline. Exemplary inhaled corticosteroids include, but are not limited to, beclomethasone, budesonide, flunisolide, fluticasone, mometasone, and triamcinolone. Exemplary systemic corticosteroids include, but are not limited to, methylprednisolone, prednisolone, and prednisone.

In some embodiments, methods of monitoring COPD and/or treatment of COPD are provided. In some embodiments, the present methods of predicting whether lung function will decline in a subject with COPD or at risk of developing COPD, and/or whether COPD will progress in a subject with COPD, and/or whether a subject will develop progressive COPD are carried out at a time 0. In some embodiments, the method is carried out again at a time 1, and optionally, a time 2, and optionally, a time 3, etc., in order to monitor the progression of COPD and/or the decline in lung function; or to monitor the effectiveness of one or more treatments of COPD at slowing the progression of COPD and/or reducing lung function decline.

EXAMPLES

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the application as defined by the appended claims. Routine molecular biology techniques described in the following examples can be carried out as described in standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2001).

Example 1 COPD Study Subjects

Subject Samples

The samples used for identifying biomarkers were from a nested case-control study from the control group of a large CT lung cancer screening study performed at University of Pittsburgh. Serum was collected at baseline (TP1) and at up to five other time points (TP2 to TP6) during follow up for several years. Not all subjects had blood samples at all 6 time points but pulmonary function tests (FEV 1%) were available for all patients at all time points at which blood samples were taken from that patient. A total of 304 samples were collected from 61 COPD patients. COPD diagnosis was determined using the GOLD (global initiative for chronic obstructive lung disease) standard. See, e.g., Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: Updated 2007. Global Initiative for Chronic Obstructive Lung Disease (GOLD); available at www.goldcopd.org. Discovery of biomarkers was performed on the baseline samples (TP1) and the subsequent longitudinal samples were used as the blinded verification set.

Sample Collection Methods

Samples were collected in red top serum tubes and processed per protocol; briefly, a sample was allowed to clot for 30 minutes at room temperature, and then centrifuged at 1300×g for 10 minutes and the top layer was removed and stored at −80° C. Samples were thawed once for aliquoting and once for the assay.

Study Inclusion and Exclusion Criteria

The inclusion criteria for subjects in the study were:

-   -   Participation in the University of Pittsburgh Medical Center         lung cancer screening program with institutional review board         approval for sample collection for the purpose of biomarker         discovery     -   Male or female, >50 years of age     -   Smokers or ex-smokers with 10 or more pack year smoking history     -   FEV1% predicted between 110 and 65 at baseline     -   At least 4 samples and timepoints     -   FEV1 at all timepoints     -   Written informed consent

The exclusion criteria for subjects in the study were:

-   -   Detected lung cancer at any time point     -   Biopsy of lung lesion at any time point         Statistical Analyses of Study Populations for Prognosis of COPD

In order to identify biomarkers that will predict whether an individual's lung function will decline or remain stable over time, the study subjects were divided into progressor or non-progressor groups. Subjects were classified as progressors if the linear regression slope was ≦−0.005 FEV1%/day, and non-progressors if the linear regression slope was >−0.005 FEV1%/day. See FIG. 1.

Subject Demographics

Table 1 shows demographic information for the study subjects at time point 1 (TP1), grouped into progressors and non-progressors according to the criteria described above.

TABLE 1 Study subject demographics Non-progressors Progressors No. of subjects 36 25 Age at PFT (yrs) 66 ± 6  63 ± 7  Male 34 23 Female 2 2 Current smokers 17 18 Ex Smokers 19 7 FEV1% 82 ± 11 85 ± 13 Smoking (PKY) 63 ± 25 64 ± 24 Data presented as median ± SD PFT = pulmonary function test PKY = pack years

As shown in Table 1, there was no significant difference in lung function, as measured by FEV1%, between the non-progressor participants and progressor participants at the onset of the study. Similarly, the two groups matched for smoking history and age.

Sample Collection Time Frame

Table 2 shows the timeframe and number of blood samples drawn at different time points (TP) for progressors and non-progressors. The blood was drawn at each follow up visit, and there was an initial 2 year interval before the first follow-up visit, hence the wide range of days in each time point. There was almost 100% compliance in follow up visit up to the 4th time point.

TABLE 2 Study subject demographics TP 2 TP 3 TP 4 TP 5 TP 6 834- 1226- 1569- 1965- 2317- TP 1 1644 2138 2351 2785 3024 Group 0 days days days days days days Total Non- 36 36 36 34 29 14 185 progressors Progressors 25 25 25 22 15 7 119 Total 61 61 61 56 44 21 304

Example 2 Multiplex Aptamer Assay for Biomarker Identification

The sample quality of the COPD samples in the two groups mentioned above were assessed by comparing distributions of markers associated with cell lysis and complement activation in the cases and controls. The sample quality was good, and there was no case control bias.

A multiplex aptamer assay was used to analyze the samples and controls to identify biomarkers predictive of progressive or non-progressive COPD. The multiplexed analysis used in this experiment included aptamers to detect 1001 proteins in blood from small sample volumes (˜8 μl of serum or plasma), with low limits of detection (1 pM median), ˜7 logs of dynamic range, and ˜5% median coefficient of variation. The multiplex aptamer assay is described, e.g., in Gold et al. (2010) Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery. PLoS ONE 5(12): e15004; and U.S. Publication Nos: 2012/0101002 and 2012/0077695.

Candidate biomarkers were identified in three different ways to create a “master list,” which was then used as the substrate for the random forest classifier model. See, e.g., Shi et al., J. Comput. Graph. Stat. 15(1): 118-138 (2006). Briefly, a random forest predictor is an ensemble of individual classification tree predictors. See, e.g., Breiman, Machine Learning, 45(1): 5-32 (2001). For each observation, each individual tree votes for one class and the forest predicts the class that has the plurality of votes. The user specifies the number of randomly selected variables (mtry) to be searched through for the best split at each node. The Gini index is used as the splitting criterion. See, e.g., Breiman et al., Classification and Regression Trees, Chapman and Hall, New York, 1984. The largest tree possible is grown and is not pruned. The root node of each tree in the forest contains a bootstrap sample from the original data as the training set. The observations that are not in the training set, roughly ⅓ of the original data set, are referred to as out-of-bag (OOB) observations. One can arrive at OOB predictions as follows: for a case in the original data, predict the outcome by plurality vote involving only those trees that did not contain the case in their corresponding bootstrap sample. By contrasting these OOB predictions with the training set outcomes, one can arrive at an estimate of the prediction error rate, which is referred to as the OOB error rate.

Univariate analysis was performed using the non-parametric Kolmogorov-Smirnov test (KS statistics), which quantifies the distance between the cumulative distribution function of each aptamer for two reference distributions designated case (progressors) and control (non-progressors). The performance of the random forest classifier is dependent upon the number and quality of the biomarkers used to construct and train the classifier. A single biomarker will perform in accordance with its KS-distance and its PCA (principal component analysis) value as exemplified herein. If a classifier performance metric is defined as the sum of the sensitivity (fraction of true positives, f_(TP)) and specificity (one minus the fraction of false positives, 1−f_(FP)), a perfect classifier will have a score of two and a random classifier, on average, will have a score of one. Using the definition of the KS-distance, that value x* which maximizes the difference in the cdf (cumulative distribution function) functions can be found by solving

$\frac{\partial{KS}}{\partial x} = {\frac{\partial\left( {{{cdf}_{c}(x)} - {{cdf}_{d}(x)}} \right)}{\partial x} = 0}$ for x which leads to p(x*|c)=p(x*|d), i.e., the KS distance occurs where the class-dependent pdfs (probability density functions) cross. Substituting this value of x* into the expression for the KS-distance yields the following definition for KS

$\begin{matrix} {{KS} = {{{cdf}_{c}\left( x^{*} \right)} - {{cdf}_{d}\left( x^{*} \right)}}} \\ {= {{\int_{- \infty}^{x^{*}}{{p\left( {x❘c} \right)}\ {\mathbb{d}x}}} - {\int_{- \infty}^{x^{*}}{{p\left( {x❘d} \right)}\ {\mathbb{d}x}}}}} \\ {= {1 - {\int_{x^{*}}^{\infty}{{p\left( {x❘c} \right)}\ {\mathbb{d}x}}} - {\int_{- \infty}^{x^{*}}{{p\left( {x❘d} \right)}\ {\mathbb{d}x}}}}} \\ {{= {1 - f_{FP} - f_{FN}}},} \end{matrix}$ the KS distance is one minus the total fraction of errors using a test with a cut-off at x*, essentially a single analyte Bayesian classifier. Since we define a score of sensitivit y+specificit y=2−f_(FP)−f_(FN), combining the above definition of the KS-distance we see that sensitivit y+specificit y=1+KS. We select biomarkers with a statistic that is inherently suited for building classifiers.

The addition of subsequent markers with good KS distances (>0.3, for example) will, in general, improve the classification performance if the subsequently added markers are independent of the first marker. Using the sensitivity plus specificity as a classifier score, many high scoring classifiers may be generated.

The three different methods for identifying candidate biomarkers were:

-   -   1. A univariate binary prognostic marker analysis to identify         biomarkers associated with decrease in lung function over time         was conducted by comparing progressors vs. non-progressors with         respect to FEV1% and generating KS-distances for each individual         marker as well as the associated p-values corrected for multiple         comparisons.     -   2. A within subject prognostic analysis to identify rate of         biomarker increase/decrease that correlated with worsening of         lung function. This was conducted by comparing the slope for a         given protein (coefficient of linear regression of RFU on time         in days) for each individual (N=61) with the individual slope in         FEV1% (coefficient of linear regression of FEV1% on time in         days) (N=61) using Spearman rho non-parametric correlation. This         process was repeated for each aptamer in the menu to create a         list of top biomarkers.     -   3. A within subject analysis to identify biomarkers at baseline         (current state RFU) that change with the rate of FEV1% (lung         function decline) over time. This correlation was repeated for         each aptamer in the menu. This analysis is similar to above,         except the baseline state of an individual is correlated with         lung function decline, rather than a protein slope correlated         with lung function decline.

The top 20 biomarkers with highest corrected p-values from each of the three comparisons listed above were combined, resulting in a set of 49 unique candidate biomarkers. A series of random forest models were then generated using backwards selection. An exemplary set of ROC plots for this analysis are shown in FIG. 2. The biomarkers included in each of the marker sets for the ROC plots in FIG. 2 are shown in Table 3, along with the AUC for each set.

TABLE 3 Markers in each set shown in FIG. 2. Markers in set AUC 1 SIGLEC7 0.77 +/− 0.14 2 SIGLEC7 CCL5 0.84 +/− 0.1  3 SIGLEC7 CCL5 IGFBP2 0.88 +/− 0.09 4 SIGLEC7 CCL5 IGFBP2 IGFBP1 0.87 +/− 0.09 5 SIGLEC7 CCL5 IGFBP2 IGFBP1 LTA 0.90 +/− 0.07 LTB 6 SIGLEC7 CCL5 IGFBP2 IGFBP1 LTA PSPN 0.87 +/− 0.09 LTB 7 SIGLEC7 CCL5 IGFBP2 IGFBP1 LTA PSPN GSK3B 0.88 +/− 0.09 LTB 8 SIGLEC7 CCL5 IGFBP2 IGFBP1 LTA PSPN GSK3B GPC2 0.86 +/− 0.09 LTB 9 SIGLEC7 CCL5 IGFBP2 IGFBP1 LTA PSPN GSK3B GPC2 LGMN 0.85 +/− 0.09 LTB

The performance of the marker sets was stable from three markers to nine markers, and the two marker set performed nearly as well as the larger sets.

Table 4 shows the top biomarkers. Table 4 also provides an alternate name for certain biomarkers, the Entrez GeneID, and the UniProt accession number for each biomarker, and whether the biomarker is present at higher or lower levels in the progressor population, as compared to the non-progressor population.

TABLE 4 Top biomarkers Biomarker level higher/lower Entrez UniProt in progressor Biomarker Gene ID ID population ANGPT2; 285 O15123 Higher Angiopoietin-2 CCDC80; URB 151887 Q76M96 Higher CCL22; MDC 6367 O00626 Higher CCL5; RANTES 6352 P13501 Higher CHST15; ST4S6 51363 Q7LFX5 Lower COL18A1; endostatin 80781 P39060 Higher CXCL5; ENA-78 6374 P42830 Lower FGF19 9965 O95750 Lower FGFR2 2263 P21802 Lower GPC2 221914 Q8N158 Lower GSK3β 2932 P49841 Higher HSPA1A; HSP70 3303 P08107 Higher IGFBP1 3484 P08833 Higher IGFBP2 3485 P18065 Higher IGFBP4 3487 P22692 Higher IGFBP7 3490 Q16270 Lower LTA.LTB; 4049, P01374, Lower lymphotoxin α1/β2 4050 Q06643 LYVE1 10894 Q9Y5Y7 Higher MST1R; MSPR 4486 Q04912 Lower PIGR 5284 P01833 Higher PSPN; persephin 5623 O60542 Lower SIGLEC7 27036 Q9Y286 Higher SLPI 6590 P03973 Higher SOD2; Mn SOD 6648 P04179 Lower THPO; TPO 7066 P40225 Lower LGMN 5641 Q99538 Higher FST 10468 P19883 Higher HS6ST1 9394 O60243 Lower LGALS3; galectin-3 3958 P17931 Higher IL10 3586 P22301 Lower

FIG. 3 shows box plots for each of the biomarkers in Table 4. The black line within each box represents the median (or 50^(th) percentile) of the data points, and the box itself represents the inter-quartile range (IQR), the area encompassing data points from the 25^(th) to 75^(th) percentile. The whiskers extend to cover data points within 1.5×IQR of the top and bottom of the box.

A substantial number of models perform well for predicting the prognosis of COPD. See FIG. 2. Nonlimiting exemplary representative six- and nine-marker classifiers are shown in Table 5. The six-marker classifier comprises the shaded biomarkers in Table 5, while the nine-marker classifier comprises all of the biomarkers in Table 5. The six- and nine-marker classifiers have an AUC of 0.87+/−0.09 and 0.85+/−0.09, respectively. The P-value is the probability that the null-hypothesis (no difference) is true for the KS statistic. The Q-value represents the corrected (for false discovery) P-value calculated based on all of the proteins detected in the original multiplex analysis. KS Rank is the rank of the marker by KS-distance relative to all proteins detected in the multiplex analysis. Signed KS is the KS-distance with its direction relative to control (+ or −).

TABLE 5 Exemplary six- and nine-marker classifiers Biomarker Signed KS P-value Q-value KS Rank SIGLEC7 0.515556 0.000386 0.421904 1 IGFBP2 0.457778 0.002514 0.959212 2 IGFBP1 0.433333 0.005004 0.959212 3 GPC2 −0.40889 0.009474 0.959212 6 LGMN 0.405556 0.010577 0.959212 7 GSK3β 0.402222 0.011616 0.959212 10 LTA.LTB −0.38111 0.027521 0.959212 13 CCE5 0.377778 0.021133 0.959212 16 PSPN −0.36889 0.03607 0.959212 17

Example 3 Cross-Validation of COPD Prognosis Classifier

A five-fold cross-validation of the six-marker classifier using the 61 sample data set was performed in an effort to guard against over-fitting. Over-fitting may occur, in some instances, in complex classification models that fit the current data well but which are less consistent when asked to classify new test samples. Such inconsistencies may occur when the model was fit to idiosyncrasies (i.e. random variation and outliers) of the training data that are absent in the new test data (i.e. the model is too specific to be used as a general tool). Methods of cross-validation are intended to identify this problem by reserving some samples that are not used to train the model (note, however, that the random forest algorithm includes testing on “out of bag” samples, thus the result is already cross-validated). The 61 baseline samples were randomly divided into subgroups of 80% training and 20% blinded test samples. The training set was used to build a model and this was fixed and tested on the remaining blinded proportion of the dataset. This was repeated five times, each ⅕th block of the data set was used once as a test set, with a different subset of ⅘th of the samples used to build the model. Each iteration was forced to use the 6 markers in the classifier described in Table 5. Five ROC curves were generated for each of the 20% blinded test sets and each was given a vote and the pooled votes for all the samples were also plotted in red.

FIG. 4 shows the five ROC plots, and the pooled votes from all of the samples. These results demonstrate that the exemplary six-marker classifier performs consistently well with all test sets, suggesting that over-fitting is not an issue in this model. The AUCs of the five verification sets ranged from 0.80+/−0.30 to 0.93+/−0.16, with four of the five verification sets having an AUC of 0.89 or higher. When all the votes are pooled, the AUC is 0.88+/−0.09, which is consistent with the original six-marker set shown in FIG. 2 and Table 3.

FIG. 5 shows an overlay of the original six-marker set shown in FIG. 2 and the pooled five-fold cross-validation set from FIG. 4. The original six-marker set and the pooled five-fold cross-validation set closely agree, again suggesting that over-fitting is not an issue.

Example 4 Blinded Validation of Classifier at Other Time Points

The random forest model that was built on the first time point (training set) was fixed and then tested on each of the later (up to 5) time points in each subject (N=233). (Note that the later blood draws are not equally spaced and the later time points had fewer individuals because some subjects were lost to follow up). As shown in FIG. 6, the model accuracy when applied to “blinded” samples from other time points was similar to the training/baseline model. In that figure, index 1=time point 1, index 2=time point 2, etc. The AUC for the six time points ranged from 0.77+/−0.12 to 0.91+/−0.15.

In addition, it was found that the model prediction of whether a person was a progressor or a non-progressor was relatively consistent when using any sample taken at any time throughout the 8 year follow-up period.

Example 5 Identification of Individuals at Risk of Developing Progressive COPD

There were a small number (N=11) of subjects who did not have COPD when enrolled in the study (GOLD score of 0 at baseline). The six-maker classifier correctly predicted that six of those 11 subjects were non-progressors, and five of the 11 subjects were progressors. See FIG. 7. Baseline (TP1) samples were used for the analysis. The width of the box plot relates to the number of subjects in each group. The two groups of non COPD subjects were significantly different with KS distances of 0.8 and P=0.0476.

Example 6 Performance of Classifier when Time Points are Averaged

To test the model accuracy when multiple time points are considered, the six-marker classifier score was averaged for each individual using all time points (mean progressor vote). Briefly, using the 6-marker classifier, the probability that the model would predict an individual as a progressor was calculated, not only at baseline, but at all available time points for that individual. Thus, although the classifier was trained only on the baseline data, the future time points were acting as pseudo-verification samples. The probability values were then averaged within an individual (n time points=n votes) to obtain a mean of the probability that an individual would be classified as a progressor by the model.

As shown in FIG. 8, the performance of the model is slightly improved by averaging over multiple time points (the confidence interval box shifts to the left, meaning improved specificity). The AUC for the averaged classifier was 0.89+/−0.08, compared to the AUC of 0.87+/−0.09 for the original classifier (at TP1).

In order to evaluate how additional follow up tests may improve performance, and when the model performance plateaus, a similar analysis was performed to look at the performance of the classifier as the number of samples used to create each individual's average score was increased systematically from 1 (the baseline only model) to include all available data points (i.e., the model shown in FIG. 8). For individuals that did not have blood draws at all time points, the mean of the available time points was used in order to include the same number of individuals (N=61) in each ROC plot.

The results are shown in FIG. 9. The ROC curve performance improved slightly when three or four time points were averaged (the AUC for the baseline and average of two time points was 0.87; the AUC for the average of three time points and four time points was 0.89 and 0.90, respectively), but did not improve with additional time points included in the average (the AUC for the average of five time points and six time points was 0.89 and 0.90, respectively).

Example 7 Exemplary Biomarker Detection Using Aptamers

An exemplary method of detecting one or more biomarkers in a sample is described, e.g., in Kraemer et al., PLoS One 6(10): e26332, and is described below. Three different methods of quantification: microarray-based hybridization, a Luminex bead-based method, and qPCR, are described.

Reagents

HEPES, NaCl, KCl, EDTA, EGTA, MgCl2 and Tween-20 may be purchased, e.g., from Fisher Biosciences. Dextran sulfate sodium salt (DxSO4), nominally 8000 molecular weight, may be purchased, e.g., from AIC and is dialyzed against deionized water for at least 20 hours with one exchange. KOD EX DNA polymerase may be purchased, e.g., from VWR. Tetramethylammonium chloride and CAPSO may be purchased, e.g., from Sigma-Aldrich and streptavidin-phycoerythrin (SAPE) may be purchased, e.g., from Moss Inc. 4-(2-Aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF) may be purchased, e.g., from Gold Biotechnology. Streptavidin-coated 96-well plates may be purchased, e.g., from Thermo Scientific (Pierce Streptavidin Coated Plates HBC, clear, 96-well, product number 15500 or 15501). NHS-PEO4-biotin may be purchased, e.g., from Thermo Scientific (EZ-Link NHS-PEO4-Biotin, product number 21329), dissolved in anhydrous DMSO, and may be stored frozen in single-use aliquots. IL-8, MIP-4, Lipocalin-2, RANTES, MMP-7, and MMP-9 may be purchased, e.g., from R&D Systems. Resistin and MCP-1 may be purchased, e.g., from PeproTech, and tPA may be purchased, e.g., from VWR.

Nucleic Acids

Conventional (including amine- and biotin-substituted) oligodeoxynucleotides may be purchased, e.g., from Integrated DNA Technologies (IDT). Z-Block is a single-stranded oligodeoxynucleotide of sequence 5′-(AC-BnBn)7-AC-3′, where Bn indicates a benzyl-substituted deoxyuridine residue. Z-block may be synthesized using conventional phosphoramidite chemistry. Aptamer capture reagents may also be synthesized by conventional phosphoramidite chemistry, and may be purified, for example, on a 21.5×75 mm PRP-3 column, operating at 80° C. on a Waters Autopurification 2767 system (or Waters 600 series semi-automated system), using, for example, a timberline TL-600 or TL-150 heater and a gradient of triethylammonium bicarbonate (TEAB)/ACN to elute product. Detection is performed at 260 nm and fractions are collected across the main peak prior to pooling best fractions.

Buffers

Buffer SB18 is composed of 40 mM HEPES, 101 mM NaCl, 5 mM KCl, 5 mM MgCl2, and 0.05% (v/v) Tween 20 adjusted to pH 7.5 with NaOH. Buffer SB17 is SB18 supplemented with 1 mM trisodium EDTA. Buffer PB1 is composed of 10 mM HEPES, 101 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM trisodium EDTA and 0.05% (v/v) Tween-20 adjusted to pH 7.5 with NaOH. CAPSO elution buffer consists of 100 mM CAPSO pH 10.0 and 1 M NaCl. Neutralization buffer contains of 500 mM HEPES, 500 mM HCl, and 0.05% (v/v) Tween-20. Agilent Hybridization Buffer is a proprietary formulation that is supplied as part of a kit (Oligo aCGH/ChIP-on-chip Hybridization Kit). Agilent Wash Buffer 1 is a proprietary formulation (Oligo aCGH/ChIP-on-chip Wash Buffer 1, Agilent). Agilent Wash Buffer 2 is a proprietary formulation (Oligo aCGH/ChIP-on-chip Wash Buffer 2, Agilent). TMAC hybridization solution consists of 4.5 M tetramethylammonium chloride, 6 mM trisodium EDTA, 75 mM Tris-HCl (pH 8.0), and 0.15% (v/v) Sarkosyl. KOD buffer (10-fold concentrated) consists of 1200 mM Tris-HCl, 15 mM MgSO4, 100 mM KCl, 60 mM (NH4)2SO4, 1% v/v Triton-X 100 and 1 mg/mL BSA.

Sample Preparation

Serum (stored at −80° C. in 100 μL aliquots) is thawed in a 25° C. water bath for 10 minutes, then stored on ice prior to sample dilution. Samples are mixed by gentle vortexing for 8 seconds. A 6% serum sample solution is prepared by dilution into 0.94× SB17 supplemented with 0.6 mM MgCl2, 1 mM trisodium EGTA, 0.8 mM AEBSF, and 2 μM Z-Block. A portion of the 6% serum stock solution is diluted 10-fold in SB17 to create a 0.6% serum stock. 6% and 0.6% stocks are used, in some embodiments, to detect high- and low-abundance analytes, respectively.

Capture Reagent (Aptamer) and Streptavidin Plate Preparation

Aptamers are grouped into 2 mixes according to the relative abundance of their cognate analytes (or biomarkers). Stock concentrations are 4 nM for each aptamer, and the final concentration of each aptamer is 0.5 nM. Aptamer stock mixes are diluted 4-fold in SB17 buffer, heated to 95° C. for 5 min and cooled to 37° C. over a 15 minute period prior to use. This denaturation-renaturation cycle is intended to normalize aptamer conformer distributions and thus ensure reproducible aptamer activity in spite of variable histories. Streptavidin plates are washed twice with 150 μL buffer PB1 prior to use.

Equilibration and Plate Capture

Heat-cooled 2× Aptamer mixes (55 μL) are combined with an equal volume of 6% or 0.6% serum dilutions, producing equilibration mixes containing 3% and 0.3% serum. The plates are sealed with a Silicone Sealing Mat (Axymat Silicone sealing mat, VWR) and incubated for 1.5 h at 37° C. Equilibration mixes are then transferred to the wells of a washed 96-well streptavidin plate and further incubated on an Eppendorf Thermomixer set at 37° C., with shaking at 800 rpm, for two hours.

Manual Assay

Unless otherwise specified, liquid is removed by dumping, followed by two taps onto layered paper towels. Wash volumes are 150 μL and all shaking incubations are done on an Eppendorf Thermomixer set at 25° C., 800 rpm. Equilibration mixes are removed by pipetting, and plates are washed twice for 1 minute with buffer PB1 supplemented with 1 mM dextran sulfate and 500 μM biotin, then 4 times for 15 seconds with buffer PB1. A freshly made solution of 1 mM NHS-PEO4-biotin in buffer PB1 (150 μL/well) is added, and plates are incubated for 5 minutes with shaking. The NHS-biotin solution is removed, and plates washed 3 times with buffer PB1 supplemented with 20 mM glycine, and 3 times with buffer PB1. Eighty-five μL of buffer PB1 supplemented with 1 mM DxSO4 is then added to each well, and plates are irradiated under a BlackRay UV lamp (nominal wavelength 365 nm) at a distance of 5 cm for 20 minutes with shaking. Samples are transferred to a fresh, washed streptavidin-coated plate, or an unused well of the existing washed streptavidin plate, combining high and low sample dilution mixtures into a single well. Samples are incubated at room temperature with shaking for 10 minutes. Unadsorbed material is removed and the plates washed 8 times for 15 seconds each with buffer PB1 supplemented with 30% glycerol. Plates are then washed once with buffer PB1. Aptamers are eluted for 5 minutes at room temperature with 100 μL CAPSO elution buffer. 90 μL of the eluate is transferred to a 96-well HybAid plate and 10 μL neutralization buffer is added.

Semi-Automated Assay

Streptavidin plates bearing adsorbed equilibration mixes are placed on the deck of a BioTek EL406 plate washer, which is programmed to perform the following steps: unadsorbed material is removed by aspiration, and wells are washed 4 times with 300 μL of buffer PB1 supplemented with 1 mM dextran sulfate and 500 μM biotin. Wells are then washed 3 times with 300 μL buffer PB1. One hundred fifty μL of a freshly prepared (from a 100 mM stock in DMSO) solution of 1 mM NHS-PEO4-biotin in buffer PB1 is added. Plates are incubated for 5 minutes with shaking. Liquid is aspirated, and wells are washed 8 times with 300 μL buffer PB1 supplemented with 10 mM glycine. One hundred μL of buffer PB1 supplemented with 1 mM dextran sulfate are added. After these automated steps, plates are removed from the plate washer and placed on a thermoshaker mounted under a UV light source (BlackRay, nominal wavelength 365 nm) at a distance of 5 cm for 20 minutes. The thermoshaker is set at 800 rpm and 25° C. After 20 minutes irradiation, samples are manually transferred to a fresh, washed streptavidin plate (or to an unused well of the existing washed plate). High-abundance (3% serum+3% aptamer mix) and low-abundance reaction mixes (0.3% serum+0.3% aptamer mix) are combined into a single well at this point. This “Catch-2” plate is placed on the deck of BioTek EL406 plate washer, which is programmed to perform the following steps: the plate is incubated for 10 minutes with shaking. Liquid is aspirated, and wells are washed 21 times with 300 μL buffer PB1 supplemented with 30% glycerol. Wells are washed 5 times with 300 μL buffer PB1, and the final wash is aspirated. One hundred μL CAPSO elution buffer are added, and aptamers are eluted for 5 minutes with shaking. Following these automated steps, the plate is then removed from the deck of the plate washer, and 90 μL aliquots of the samples are transferred manually to the wells of a HybAid 96-well plate that contains 10 μL neutralization buffer.

Hybridization to Custom Agilent 8×15 k Microarrays

24 μL of the neutralized eluate is transferred to a new 96-well plate and 6 μL of 10× Agilent Block (Oligo aCGH/ChIP-on-chip Hybridization Kit, Large Volume, Agilent 5188-5380), containing a set of hybridization controls composed of 10 Cy3 aptamers is added to each well. Thirty μL 2× Agilent Hybridization buffer is added to each sample and mixed. Forty μL of the resulting hybridization solution is manually pipetted into each “well” of the hybridization gasket slide (Hybridization Gasket Slide, 8-microarray per slide format, Agilent). Custom Agilent microarray slides, bearing 10 probes per array complementary to 40 nucleotide random region of each aptamer with a 20× dT linker, are placed onto the gasket slides according to the manufacturers' protocol. The assembly (Hybridization Chamber Kit—SureHyb-enabled, Agilent) is clamped and incubated for 19 hours at 60° C. while rotating at 20 rpm.

Post Hybridization Washing

Approximately 400 mL Agilent Wash Buffer 1 is placed into each of two separate glass staining dishes. Slides (no more than two at a time) are disassembled and separated while submerged in Wash Buffer 1, then transferred to a slide rack in a second staining dish also containing Wash Buffer 1. Slides are incubated for an additional 5 minutes in Wash Buffer 1 with stirring. Slides are transferred to Wash Buffer 2 pre-equilibrated to 37° C. and incubated for 5 minutes with stirring. Slides are transferred to a fourth staining dish containing acetonitrile, and incubated for 5 minutes with stirring.

Microarray Imaging

Microarray slides are imaged with an Agilent G2565CA Microarray Scanner System, using the Cy3-channel at 5 μm resolution at 100% PMT setting, and the XRD option enabled at 0.05. The resulting TIFF images are processed using Agilent feature extraction software version 10.5.1.1 with the GE1_105_Dec08 protocol. Primary Agilent data is available as Supplementary Information (FIG. S6).

Luminex Probe Design

Probes immobilized to beads have 40 deoxynucleotides complementary to the 3′ end of the 40 nucleotide random region of the target aptamer. The aptamer complementary region is coupled to Luminex Microspheres through a hexaethyleneglycol (HEG) linker bearing a 5′ amino terminus Biotinylated detection deoxyoligonucleotides comprise 17-21 deoxynucleotides complementary to the 5′ primer region of target aptamers. Biotin moieties are appended to the 3′ ends of detection oligos.

Coupling of Probes to Luminex Microspheres

Probes are coupled to Luminex Microplex Microspheres essentially per the manufacturer's instructions, but with the following modifications: amino-terminal oligonucleotide amounts are 0.08 nMol per 2.5×106 microspheres, and the second EDC addition is 5 μL at 10 mg/mL. Coupling reactions are performed in an Eppendorf ThermoShaker set at 25° C. and 600 rpm.

Microsphere Hybridization

Microsphere stock solutions (about 40000 microspheres/μL) are vortexed and sonicated in a Health Sonics ultrasonic cleaner (Model: T1.9C) for 60 seconds to suspend the microspheres. Suspended microspheres are diluted to 2000 microspheres per reaction in 1.5× TMAC hybridization solutions and mixed by vortexing and sonication. Thirty-three μL per reaction of the bead mixture are transferred into a 96-well HybAid plate. Seven μL of 15 nM biotinylated detection oligonucleotide stock in 1× TE buffer are added to each reaction and mixed. Ten μL of neutralized assay sample are added and the plate is sealed with a silicon cap mat seal. The plate is first incubated at 96° C. for 5 minutes and incubated at 50° C. without agitation overnight in a conventional hybridization oven. A filter plate (Dura pore, Millipore part number MSBVN1250, 1.2 μm pore size) is prewetted with 75 μL 1× TMAC hybridization solution supplemented with 0.5% (w/v) BSA. The entire sample volume from the hybridization reaction is transferred to the filter plate. The hybridization plate is rinsed with 75 μL 1× TMAC hybridization solution containing 0.5% BSA and any remaining material is transferred to the filter plate. Samples are filtered under slow vacuum, with 150 μL buffer evacuated over about 8 seconds. The filter plate is washed once with 75 μL 1× TMAC hybridization solution containing 0.5% BSA and the microspheres in the filter plate are resuspended in 75 μL 1× TMAC hybridization solution containing 0.5% BSA. The filter plate is protected from light and incubated on an Eppendorf Thermalmixer R for 5 minutes at 1000 rpm. The filter plate is then washed once with 75 μL 1× TMAC hybridization solution containing 0.5% BSA. 75 μL of 10 μg/mL streptavidin phycoerythrin (SAPE-100, MOSS, Inc.) in 1× TMAC hybridization solution is added to each reaction and incubated on Eppendorf Thermalmixer R at 25° C. at 1000 rpm for 60 minutes. The filter plate is washed twice with 75 μL 1× TMAC hybridization solution containing 0.5% BSA and the microspheres in the filter plate are resuspended in 75 μL 1× TMAC hybridization solution containing 0.5% BSA. The filter plate is then incubated protected from light on an Eppendorf Thermalmixer R for 5 minutes, 1000 rpm. The filter plate is then washed once with 75 μL 1× TMAC hybridization solution containing 0.5% BSA. Microspheres are resuspended in 75 μL 1× TMAC hybridization solution supplemented with 0.5% BSA, and analyzed on a Luminex 100 instrument running XPonent 3.0 software. At least 100 microspheres are counted per bead type, under high PMT calibration and a doublet discriminator setting of 7500 to 18000.

QPCR Read-Out

Standard curves for qPCR are prepared in water ranging from 108 to 102 copies with 10-fold dilutions and a no-template control. Neutralized assay samples are diluted 40-fold into diH2O. The qPCR master mix is prepared at 2× final concentration (2× KOD buffer, 400 μM dNTP mix, 400 nM forward and reverse primer mix, 2× SYBR Green I and 0.5 U KOD EX). Ten μL of 2× qPCR master mix is added to 10 μL of diluted assay sample. qPCR is run on a BioRad MyIQ iCycler with 2 minutes at 96° C. followed by 40 cycles of 96° C. for 5 seconds and 72° C. for 30 seconds.

Example 8

A KS distance cutoff of 0.35 was used to select the top biomarkers from the original set for building a naïve B ayes classifier predicting progression (lung function decline) from any “n” markers of the set. Table 6 shows a set of selected biomarkers, along with the KS distance, log 2(fold change) (or “log 2(FC)”), p-value, and q-value for each marker.

TABLE 6 Selected biomarkers Protein KS distance log2(FC) p-Value q-Value SIGLEC7 0.515556 0.210385 0.000386 0.421904 IGFBP2 0.457778 0.514709 0.002514 0.959212 IGFBP1 0.433333 1.132172 0.005004 0.959212 PIGR 0.414444 0.523805 0.008439 0.959212 HSPA1A 0.414444 0.222205 0.008439 0.959212 GPC2 −0.40889 −0.0416 0.009474 0.959212 LGMN 0.405556 0.09267 0.010577 0.959212 ANGPT2 0.402222 0.30062 0.011616 0.959212 GSK3β 0.402222 0.070798 0.011616 0.959212 PDGFRβ 0.393333 0.340947 0.014298 0.959212 LYVE1 0.383333 0.272157 0.018383 0.959212 LTA-LTB −0.38111 −0.04211 0.027521 0.959212 IL12B-IL23A −0.38 −0.0398 0.019942 0.959212 FGF19 −0.37778 −0.14655 0.021133 0.959212 CCL5 0.377778 0.202753 0.021133 0.959212 PSPN −0.36889 −0.06276 0.03607 0.959212

All of the markers in Table 6 are also in Table 4, except for PDGFRβ and IL12B-IL23A. PDGFRP (Entrez Gene ID 5159; UniProt P09619) is elevated in progressive disease, while IL12B-IL23A (Entrez Gene ID 3593 and 51561; UniProt P29460, Q9NPF7) is reduced.

A naive Bayes classifier was built to predict progression (lung function decline) from any “n” markers (n=1,2,3), using all single, pairwise, and triplicate combinations of the biomarker proteins in Table 6. This analysis resulted in 16, 120, and 560 possible combinations (and models) for n=1, 2, and 3 respectively. Classifier performance (sensitivity+specificity) was then assessed for each model. The ‘null’ performance was produced as above, except a random protein (or proteins) was selected from the entire protein array excluding the proteins in Table 6.

FIG. 13 shows paired histograms of the null performance (random proteins) and model performance (selected biomarkers) are provided showing the single marker naive Bayes classifiers (n=1; A), the pairwise marker classifiers (n=2; B), and the triplicate marker classifiers (n=3; C). The null performance models are represented by a dotted line and the biomarker models themselves are represented by a solid line. The results in FIG. 13 demonstrate that the particular biomarkers listed in Table 6 represent a robust group of markers from which many groups of markers can be selected that will be predictive for COPD progression (lung function decline).

Tables 7, 8, and 9 show each single (Table 7), each pair (Table 8) and each triplicate (Table 9) group of biomarkers from Table 6. The sensitivity and specificity for each group, sensitivity+specificity, area under the curve (AUC), lower 95% confidence interval (lower 95% CI), and upper 95% confidence interval (upper 95% CI) are also shown in the tables.

TABLE 7 Single biomarker classifiers Sensi- Speci- Sens + Lower Upper biomarker1 tivity ficity Spec AUC 95% CI 95% CI SIGLEC7 0.6 0.778 1.378 0.797 0.684 0.91 IGFBP2 0.52 0.833 1.353 0.724 0.587 0.862 ANGPT2 0.6 0.75 1.35 0.707 0.575 0.838 IGFBP1 0.64 0.694 1.334 0.737 0.607 0.866 LTA-LTB 0.4 0.917 1.317 0.714 0.578 0.851 GPC2 0.48 0.833 1.313 0.663 0.517 0.81 CCL5 0.36 0.944 1.304 0.814 0.703 0.926 PDGFRβ 0.44 0.861 1.301 0.687 0.547 0.826 PIGR 0.48 0.806 1.286 0.708 0.569 0.847 PSPN 0.4 0.861 1.261 0.703 0.565 0.842 LGMN 0.4 0.833 1.233 0.72 0.588 0.852 GSK3B 0.4 0.833 1.233 0.672 0.532 0.813 LYVE1 0.48 0.75 1.23 0.65 0.508 0.792 FGF19 0.56 0.667 1.227 0.676 0.536 0.815 HSPA1A 0.48 0.722 1.202 0.667 0.522 0.811 IL12B- 0.32 0.639 0.959 0.601 0.456 0.746 IL23A

TABLE 8 Pairwise biomarker classifiers Lower Upper Biomark. 1 Biomark. 2 Sens. Spec. Sens + Spec AUC 95% CI 95% CI SIGLEC7 IGFBP2 0.8 0.833 1.633 0.861 0.765 0.957 SIGLEC7 IGFBP1 0.8 0.806 1.606 0.841 0.74 0.942 IGFBP1 CCL5 0.8 0.806 1.606 0.823 0.709 0.937 IL12B- CCL5 0.68 0.889 1.569 0.829 0.716 0.942 IL23A SIGLEC7 GPC2 0.76 0.806 1.566 0.786 0.664 0.907 LTA-LTB CCL5 0.72 0.833 1.553 0.856 0.759 0.952 IGFBP2 LTA-LTB 0.8 0.75 1.55 0.812 0.7 0.924 LGMN CCL5 0.68 0.861 1.541 0.811 0.702 0.92 SIGLEC7 CCL5 0.76 0.778 1.538 0.838 0.738 0.938 PIGR PDGFRβ 0.84 0.694 1.534 0.798 0.686 0.909 LTA-LTB FGF19 0.84 0.694 1.534 0.772 0.643 0.902 IGFBP2 GSK3B 0.72 0.806 1.526 0.777 0.663 0.89 IGFBP1 HSPA1A 0.8 0.722 1.522 0.78 0.658 0.902 ANGPT2 CCL5 0.68 0.833 1.513 0.826 0.717 0.934 IGFBP1 LGMN 0.84 0.667 1.507 0.79 0.676 0.904 SIGLEC7 PDGFRβ 0.72 0.778 1.498 0.794 0.679 0.909 LGMN PSPN 0.72 0.778 1.498 0.769 0.646 0.892 SIGLEC7 LGMN 0.8 0.694 1.494 0.829 0.728 0.93 IGFBP2 LGMN 0.8 0.694 1.494 0.811 0.7 0.922 ANGPT2 PSPN 0.8 0.694 1.494 0.799 0.687 0.911 CCL5 PSPN 0.68 0.806 1.486 0.794 0.676 0.913 SIGLEC7 PIGR 0.76 0.722 1.482 0.799 0.687 0.911 SIGLEC7 PSPN 0.76 0.722 1.482 0.811 0.698 0.924 IGFBP1 IL12B- 0.76 0.722 1.482 0.763 0.643 0.884 IL23A ANGPT2 LTA-LTB 0.76 0.722 1.482 0.777 0.662 0.892 IGFBP1 LTA-LTB 0.84 0.639 1.479 0.781 0.664 0.898 LGMN LYVE1 0.84 0.639 1.479 0.752 0.626 0.878 SIGLEC7 LTA-LTB 0.72 0.75 1.47 0.824 0.719 0.93 SIGLEC7 FGF19 0.72 0.75 1.47 0.806 0.69 0.921 IGFBP1 GPC2 0.72 0.75 1.47 0.736 0.601 0.87 IGFBP1 ANGPT2 0.72 0.75 1.47 0.779 0.659 0.899 ANGPT2 FGF19 0.72 0.75 1.47 0.746 0.613 0.878 LYVE1 CCL5 0.72 0.75 1.47 0.801 0.68 0.922 FGF19 CCL5 0.72 0.75 1.47 0.786 0.66 0.911 ANGPT2 IL12B- 0.8 0.667 1.467 0.759 0.636 0.882 IL23A IL12B- PSPN 0.8 0.667 1.467 0.767 0.648 0.885 IL23A GPC2 LGMN 0.68 0.778 1.458 0.734 0.597 0.872 GPC2 LYVE1 0.68 0.778 1.458 0.714 0.574 0.855 LGMN LTA-LTB 0.68 0.778 1.458 0.782 0.658 0.906 SIGLEC7 IL12B- 0.76 0.694 1.454 0.842 0.744 0.941 IL23A IGFBP2 PDGFRβ 0.76 0.694 1.454 0.803 0.688 0.919 IGFBP1 PIGR 0.76 0.694 1.454 0.77 0.652 0.888 IGFBP1 FGF19 0.76 0.694 1.454 0.723 0.592 0.855 PIGR PSPN 0.76 0.694 1.454 0.729 0.598 0.86 HSPA1A LYVE1 0.76 0.694 1.454 0.741 0.607 0.875 IGFBP2 CCL5 0.64 0.806 1.446 0.81 0.697 0.923 IGFBP2 ANGPT2 0.72 0.722 1.442 0.776 0.655 0.896 PIGR LTA-LTB 0.68 0.75 1.43 0.768 0.64 0.895 PIGR CCL5 0.68 0.75 1.43 0.8 0.69 0.91 LGMN ANGPT2 0.68 0.75 1.43 0.766 0.646 0.885 PDGFRβ CCL5 0.68 0.75 1.43 0.811 0.699 0.923 PIGR HSPA1A 0.76 0.667 1.427 0.752 0.625 0.879 SIGLEC7 GSK3B 0.64 0.778 1.418 0.809 0.708 0.909 PIGR GSK3B 0.64 0.778 1.418 0.767 0.648 0.885 GPC2 LTA-LTB 0.64 0.778 1.418 0.721 0.58 0.862 PDGFRβ PSPN 0.64 0.778 1.418 0.736 0.608 0.863 IGFBP1 LYVE1 0.72 0.694 1.414 0.762 0.636 0.888 PIGR ANGPT2 0.72 0.694 1.414 0.766 0.643 0.889 PDGFRβ FGF19 0.72 0.694 1.414 0.76 0.634 0.886 HSPA1A IL12B- 0.8 0.611 1.411 0.721 0.591 0.852 IL23A GPC2 CCL5 0.6 0.806 1.406 0.726 0.587 0.864 PDGFRβ LTA-LTB 0.6 0.806 1.406 0.701 0.56 0.842 LTA-LTB PSPN 0.6 0.806 1.406 0.752 0.625 0.879 SIGLEC7 HSPA1A 0.68 0.722 1.402 0.763 0.641 0.885 SIGLEC7 ANGPT2 0.68 0.722 1.402 0.791 0.678 0.904 SIGLEC7 LYVE1 0.68 0.722 1.402 0.783 0.665 0.902 GPC2 PSPN 0.68 0.722 1.402 0.719 0.579 0.859 LGMN PDGFRβ 0.68 0.722 1.402 0.783 0.664 0.903 ANGPT2 PDGFRβ 0.68 0.722 1.402 0.788 0.67 0.906 LYVE1 FGF19 0.68 0.722 1.402 0.708 0.569 0.847 ANGPT2 LYVE1 0.76 0.639 1.399 0.718 0.585 0.85 IL12B- FGF19 0.76 0.639 1.399 0.716 0.582 0.849 IL23A FGF19 PSPN 0.84 0.556 1.396 0.751 0.619 0.883 PIGR GPC2 0.64 0.75 1.39 0.752 0.62 0.884 LYVE1 LTA-LTB 0.72 0.667 1.387 0.751 0.628 0.874 LYVE1 PSPN 0.72 0.667 1.387 0.726 0.592 0.859 IGFBP2 HSPA1A 0.68 0.694 1.374 0.744 0.62 0.869 IGFBP2 IL12B- 0.68 0.694 1.374 0.75 0.627 0.873 IL23A IGFBP2 PSPN 0.68 0.694 1.374 0.786 0.669 0.902 HSPA1A GPC2 0.68 0.694 1.374 0.719 0.584 0.854 HSPA1A CCL5 0.68 0.694 1.374 0.742 0.615 0.869 LYVE1 IL12B- 0.76 0.611 1.371 0.73 0.601 0.859 IL23A PIGR LYVE1 0.72 0.639 1.359 0.753 0.629 0.877 IGFBP1 PDGFRβ 0.68 0.667 1.347 0.747 0.618 0.876 IGFBP1 PSPN 0.68 0.667 1.347 0.742 0.612 0.873 HSPA1A LTA-LTB 0.68 0.667 1.347 0.759 0.635 0.883 IGFBP2 PIGR 0.64 0.694 1.334 0.746 0.615 0.876 PIGR LGMN 0.64 0.694 1.334 0.768 0.644 0.892 LGMN IL12B- 0.64 0.694 1.334 0.739 0.613 0.864 IL23A LTA-LTB IL12B- 0.64 0.694 1.334 0.794 0.682 0.907 IL23A IGFBP2 IGFBP1 0.72 0.611 1.331 0.761 0.637 0.886 PIGR FGF19 0.72 0.611 1.331 0.734 0.602 0.867 GSK3B CCL5 0.52 0.806 1.326 0.803 0.703 0.904 PIGR IL12B- 0.6 0.722 1.322 0.751 0.63 0.872 IL23A HSPA1A GSK3B 0.6 0.722 1.322 0.75 0.635 0.865 HSPA1A FGF19 0.68 0.639 1.319 0.701 0.564 0.838 GPC2 IL12B- 0.68 0.639 1.319 0.68 0.541 0.819 IL23A LGMN FGF19 0.68 0.639 1.319 0.759 0.626 0.892 IGFBP2 GPC2 0.56 0.75 1.31 0.738 0.608 0.867 IGFBP2 LYVE1 0.64 0.667 1.307 0.76 0.632 0.888 IGFBP2 FGF19 0.64 0.667 1.307 0.732 0.602 0.863 HSPA1A PDGFRβ 0.72 0.583 1.303 0.721 0.592 0.85 HSPA1A PSPN 0.72 0.583 1.303 0.713 0.581 0.845 IGFBP1 GSK3B 0.44 0.861 1.301 0.726 0.603 0.848 GSK3B IL12B- 0.44 0.861 1.301 0.719 0.6 0.838 IL23A LGMN GSK3B 0.52 0.778 1.298 0.758 0.644 0.872 GSK3B LTA-LTB 0.52 0.778 1.298 0.748 0.632 0.864 GSK3B PSPN 0.52 0.778 1.298 0.729 0.611 0.847 HSPA1A ANGPT2 0.68 0.611 1.291 0.698 0.564 0.832 GSK3B LYVE1 0.48 0.806 1.286 0.753 0.641 0.866 GSK3B FGF19 0.48 0.806 1.286 0.732 0.607 0.857 GPC2 FGF19 0.64 0.639 1.279 0.702 0.564 0.84 GPC2 GSK3B 0.44 0.806 1.246 0.716 0.585 0.846 ANGPT2 GSK3B 0.44 0.806 1.246 0.758 0.645 0.87 HSPA1A LGMN 0.6 0.639 1.239 0.718 0.583 0.852 GPC2 ANGPT2 0.6 0.639 1.239 0.711 0.578 0.844 PDGFRβ LYVE1 0.56 0.667 1.227 0.72 0.592 0.848 GPC2 PDGFRβ 0.52 0.694 1.214 0.688 0.548 0.828 GSK3B PDGFRβ 0.44 0.75 1.19 0.721 0.593 0.85 PDGFRβ IL12B- 0.6 0.583 1.183 0.728 0.601 0.855 IL23A

TABLE 9 Triplicate biomarker classifiers Lower Upper Biom. 1 Biom. 2 Biom. 3 Sens. Spec. Sens + Spec AUC 95% CI 95% CI ANGPT2 LTA-LTB CCL5 0.88 0.833 1.713 0.868 0.773 0.963 LTA-LTB IL12B- CCL5 0.84 0.861 1.701 0.893 0.809 0.978 IL23A SIGLEC7 IGFBP2 PDGFRβ 0.88 0.806 1.686 0.848 0.744 0.952 SIGLEC7 IGFBP2 LTA-LTB 0.84 0.833 1.673 0.878 0.788 0.968 SIGLEC7 IGFBP2 CCL5 0.84 0.833 1.673 0.874 0.784 0.965 SIGLEC7 IGFBP2 IL12B- 0.8 0.861 1.661 0.896 0.811 0.98 IL23A SIGLEC7 IGFBP2 PIGR 0.88 0.778 1.658 0.844 0.746 0.943 SIGLEC7 IGFBP2 LGMN 0.84 0.806 1.646 0.898 0.817 0.979 IGFBP1 LTA-LTB CCL5 0.84 0.806 1.646 0.859 0.764 0.953 ANGPT2 PDGFRβ FGF19 0.84 0.806 1.646 0.819 0.701 0.937 PDGFRβ FGF19 CCL5 0.84 0.806 1.646 0.816 0.695 0.937 IGFBP2 LGMN PDGFRβ 0.92 0.722 1.642 0.87 0.771 0.969 IGFBP2 LGMN LTA-LTB 0.92 0.722 1.642 0.857 0.759 0.954 SIGLEC7 IGFBP2 PSPN 0.8 0.833 1.633 0.848 0.741 0.954 ANGPT2 PDGFRβ CCL5 0.8 0.833 1.633 0.834 0.729 0.94 PIGR PDGFRβ IL12B- 0.88 0.75 1.63 0.84 0.739 0.941 IL23A SIGLEC7 IGFBP2 GPC2 0.76 0.861 1.621 0.838 0.727 0.948 SIGLEC7 IGFBP1 IL12B- 0.76 0.861 1.621 0.871 0.783 0.96 IL23A IGFBP1 CCL5 PSPN 0.76 0.861 1.621 0.838 0.733 0.942 SIGLEC7 IGFBP1 LGMN 0.84 0.778 1.618 0.9 0.821 0.979 SIGLEC7 LTA-LTB CCL5 0.84 0.778 1.618 0.852 0.757 0.947 IGFBP2 GSK3B LTA-LTB 0.84 0.778 1.618 0.812 0.709 0.916 IGFBP1 LGMN CCL5 0.84 0.778 1.618 0.874 0.784 0.965 IGFBP1 LYVE1 IL12B- 0.84 0.778 1.618 0.802 0.685 0.919 IL23A LYVE1 IL12B- CCL5 0.84 0.778 1.618 0.84 0.729 0.951 IL23A IGFBP2 PDGFRβ LTA-LTB 0.8 0.806 1.606 0.833 0.727 0.939 ANGPT2 IL12B- CCL5 0.8 0.806 1.606 0.85 0.745 0.955 IL23A LTA-LTB FGF19 CCL5 0.8 0.806 1.606 0.817 0.695 0.938 FGF19 CCL5 PSPN 0.8 0.806 1.606 0.813 0.688 0.939 IGFBP1 LTA-LTB IL12B- 0.88 0.722 1.602 0.839 0.737 0.941 IL23A ANGPT2 IL12B- PSPN 0.88 0.722 1.602 0.826 0.718 0.933 IL23A SIGLEC7 IGFBP2 IGFBP1 0.76 0.833 1.593 0.861 0.761 0.961 SIGLEC7 IGFBP2 FGF19 0.76 0.833 1.593 0.853 0.745 0.961 ANGPT2 CCL5 PSPN 0.76 0.833 1.593 0.856 0.753 0.958 SIGLEC7 PIGR CCL5 0.84 0.75 1.59 0.846 0.748 0.943 SIGLEC7 HSPA1A LTA-LTB 0.84 0.75 1.59 0.8 0.686 0.914 IGFBP2 PIGR PDGFRβ 0.84 0.75 1.59 0.839 0.74 0.938 IGFBP2 PDGFRβ CCL5 0.84 0.75 1.59 0.848 0.747 0.948 IGFBP1 ANGPT2 LYVE1 0.84 0.75 1.59 0.816 0.703 0.929 SIGLEC7 IGFBP1 CCL5 0.8 0.778 1.578 0.898 0.824 0.972 SIGLEC7 PIGR IL12B- 0.8 0.778 1.578 0.829 0.727 0.931 IL23A SIGLEC7 LGMN CCL5 0.8 0.778 1.578 0.874 0.788 0.961 IGFBP2 LGMN GSK3B 0.8 0.778 1.578 0.823 0.726 0.92 IGFBP2 PDGFRβ IL12B- 0.8 0.778 1.578 0.838 0.732 0.943 IL23A IGFBP2 CCL5 PSPN 0.8 0.778 1.578 0.816 0.704 0.927 IGFBP1 HSPA1A GPC2 0.8 0.778 1.578 0.776 0.648 0.903 IGFBP1 HSPA1A CCL5 0.8 0.778 1.578 0.809 0.695 0.923 IGFBP1 ANGPT2 IL12B- 0.8 0.778 1.578 0.802 0.686 0.918 IL23A LGMN LTA-LTB FGF19 0.8 0.778 1.578 0.811 0.684 0.938 LTA-LTB CCL5 PSPN 0.8 0.778 1.578 0.821 0.715 0.927 IL12B- CCL5 PSPN 0.8 0.778 1.578 0.844 0.742 0.947 IL23A PIGR PDGFRβ FGF19 0.88 0.694 1.574 0.804 0.683 0.926 LGMN LYVE1 IL12B- 0.88 0.694 1.574 0.78 0.66 0.9 IL23A SIGLEC7 ANGPT2 CCL5 0.76 0.806 1.566 0.83 0.728 0.932 SIGLEC7 LTA-LTB PSPN 0.76 0.806 1.566 0.828 0.72 0.936 SIGLEC7 IL12B- FGF19 0.76 0.806 1.566 0.828 0.716 0.939 IL23A SIGLEC7 IL12B- CCL5 0.76 0.806 1.566 0.877 0.79 0.963 IL23A PIGR LTA-LTB CCL5 0.76 0.806 1.566 0.827 0.723 0.93 GPC2 LGMN LYVE1 0.76 0.806 1.566 0.764 0.628 0.901 LGMN ANGPT2 FGF19 0.76 0.806 1.566 0.771 0.642 0.9 LGMN CCL5 PSPN 0.76 0.806 1.566 0.81 0.697 0.923 ANGPT2 LYVE1 FGF19 0.76 0.806 1.566 0.758 0.625 0.891 SIGLEC7 PIGR PDGFRβ 0.84 0.722 1.562 0.831 0.729 0.933 SIGLEC7 LTA-LTB IL12B- 0.84 0.722 1.562 0.873 0.782 0.965 IL23A IGFBP2 PDGFRβ LYVE1 0.84 0.722 1.562 0.821 0.708 0.934 IGFBP1 HSPA1A LYVE1 0.84 0.722 1.562 0.777 0.652 0.901 IGFBP1 ANGPT2 PDGFRβ 0.84 0.722 1.562 0.807 0.691 0.922 HSPA1A LYVE1 PSPN 0.84 0.722 1.562 0.753 0.621 0.885 IGFBP2 LYVE1 CCL5 0.72 0.833 1.553 0.824 0.706 0.943 PIGR FGF19 CCL5 0.72 0.833 1.553 0.777 0.652 0.901 GPC2 LTA-LTB CCL5 0.72 0.833 1.553 0.762 0.631 0.893 SIGLEC7 IGFBP2 ANGPT2 0.8 0.75 1.55 0.848 0.751 0.944 SIGLEC7 IGFBP2 GSK3B 0.8 0.75 1.55 0.834 0.738 0.931 SIGLEC7 HSPA1A PSPN 0.8 0.75 1.55 0.777 0.656 0.897 SIGLEC7 ANGPT2 PSPN 0.8 0.75 1.55 0.821 0.714 0.928 SIGLEC7 IL12B- PSPN 0.8 0.75 1.55 0.846 0.744 0.948 IL23A IGFBP2 IGFBP1 GPC2 0.8 0.75 1.55 0.764 0.636 0.893 IGFBP2 IGFBP1 GSK3B 0.8 0.75 1.55 0.768 0.65 0.885 IGFBP2 IGFBP1 PDGFRβ 0.8 0.75 1.55 0.796 0.675 0.917 IGFBP2 PIGR HSPA1A 0.8 0.75 1.55 0.77 0.644 0.896 IGFBP2 LGMN CCL5 0.8 0.75 1.55 0.862 0.767 0.958 IGFBP2 ANGPT2 PSPN 0.8 0.75 1.55 0.808 0.696 0.919 IGFBP2 PDGFRβ PSPN 0.8 0.75 1.55 0.804 0.691 0.918 IGFBP2 LTA-LTB CCL5 0.8 0.75 1.55 0.86 0.766 0.954 IGFBP1 PIGR PDGFRβ 0.8 0.75 1.55 0.816 0.708 0.924 IGFBP1 HSPA1A LGMN 0.8 0.75 1.55 0.808 0.693 0.922 IGFBP1 LGMN LYVE1 0.8 0.75 1.55 0.821 0.714 0.929 IGFBP1 ANGPT2 CCL5 0.8 0.75 1.55 0.852 0.754 0.95 IGFBP1 LYVE1 LTA-LTB 0.8 0.75 1.55 0.824 0.72 0.929 PIGR ANGPT2 PDGFRβ 0.8 0.75 1.55 0.847 0.748 0.945 HSPA1A LTA-LTB IL12B- 0.8 0.75 1.55 0.804 0.692 0.917 IL23A LYVE1 LTA-LTB CCL5 0.8 0.75 1.55 0.838 0.736 0.94 IGFBP2 HSPA1A PDGFRβ 0.88 0.667 1.547 0.799 0.687 0.911 IGFBP1 LGMN LTA-LTB 0.88 0.667 1.547 0.856 0.763 0.948 PIGR LGMN PDGFRβ 0.88 0.667 1.547 0.829 0.724 0.933 PIGR PDGFRβ CCL5 0.88 0.667 1.547 0.824 0.722 0.927 IGFBP2 IL12B- CCL5 0.68 0.861 1.541 0.812 0.703 0.921 IL23A IGFBP2 FGF19 CCL5 0.68 0.861 1.541 0.797 0.678 0.916 LGMN FGF19 CCL5 0.68 0.861 1.541 0.803 0.679 0.927 SIGLEC7 IGFBP1 HSPA1A 0.76 0.778 1.538 0.821 0.713 0.929 SIGLEC7 HSPA1A IL12B- 0.76 0.778 1.538 0.79 0.673 0.907 IL23A SIGLEC7 GPC2 LGMN 0.76 0.778 1.538 0.816 0.704 0.927 SIGLEC7 LYVE1 CCL5 0.76 0.778 1.538 0.813 0.703 0.924 SIGLEC7 LTA-LTB FGF19 0.76 0.778 1.538 0.826 0.712 0.939 SIGLEC7 CCL5 PSPN 0.76 0.778 1.538 0.838 0.735 0.94 IGFBP2 GPC2 LGMN 0.76 0.778 1.538 0.793 0.675 0.912 IGFBP1 PIGR GPC2 0.76 0.778 1.538 0.773 0.649 0.898 IGFBP1 PIGR LGMN 0.76 0.778 1.538 0.823 0.72 0.927 IGFBP1 HSPA1A LTA-LTB 0.76 0.778 1.538 0.81 0.7 0.92 IGFBP1 PDGFRβ CCL5 0.76 0.778 1.538 0.824 0.715 0.934 HSPA1A GPC2 LYVE1 0.76 0.778 1.538 0.761 0.628 0.894 HSPA1A GPC2 LTA-LTB 0.76 0.778 1.538 0.759 0.628 0.889 GPC2 LGMN LTA-LTB 0.76 0.778 1.538 0.777 0.644 0.909 LGMN PDGFRβ FGF19 0.76 0.778 1.538 0.808 0.687 0.929 LGMN LTA-LTB CCL5 0.76 0.778 1.538 0.858 0.766 0.949 ANGPT2 LTA-LTB PSPN 0.76 0.778 1.538 0.844 0.745 0.944 PDGFRβ IL12B- CCL5 0.76 0.778 1.538 0.846 0.746 0.945 IL23A LYVE1 CCL5 PSPN 0.76 0.778 1.538 0.783 0.659 0.908 SIGLEC7 PDGFRβ IL12B- 0.84 0.694 1.534 0.841 0.739 0.943 IL23A SIGLEC7 LYVE1 LTA-LTB 0.84 0.694 1.534 0.827 0.72 0.933 IGFBP2 LGMN LYVE1 0.84 0.694 1.534 0.833 0.727 0.94 IGFBP2 ANGPT2 PDGFRβ 0.84 0.694 1.534 0.839 0.733 0.945 IGFBP2 LYVE1 LTA-LTB 0.84 0.694 1.534 0.843 0.745 0.942 IGFBP1 LGMN ANGPT2 0.84 0.694 1.534 0.832 0.728 0.937 IGFBP1 PDGFRβ IL12B- 0.84 0.694 1.534 0.803 0.689 0.918 IL23A PIGR HSPA1A LGMN 0.84 0.694 1.534 0.784 0.664 0.905 PIGR HSPA1A LTA-LTB 0.84 0.694 1.534 0.781 0.662 0.9 PIGR PDGFRβ LYVE1 0.84 0.694 1.534 0.818 0.709 0.927 PIGR LYVE1 LTA-LTB 0.84 0.694 1.534 0.819 0.71 0.928 HSPA1A LYVE1 LTA-LTB 0.84 0.694 1.534 0.79 0.672 0.908 HSPA1A LTA-LTB FGF19 0.84 0.694 1.534 0.763 0.635 0.892 LGMN ANGPT2 LYVE1 0.84 0.694 1.534 0.789 0.67 0.908 LGMN FGF19 PSPN 0.84 0.694 1.534 0.809 0.682 0.936 ANGPT2 PDGFRβ IL12B- 0.84 0.694 1.534 0.842 0.743 0.941 IL23A LTA-LTB IL12B- FGF19 0.84 0.694 1.534 0.814 0.697 0.932 IL23A PIGR HSPA1A PDGFRβ 0.92 0.611 1.531 0.807 0.694 0.919 SIGLEC7 IGFBP1 ANGPT2 0.72 0.806 1.526 0.861 0.77 0.952 IGFBP2 IGFBP1 CCL5 0.72 0.806 1.526 0.788 0.672 0.904 IGFBP2 GSK3B IL12B- 0.72 0.806 1.526 0.798 0.692 0.903 IL23A IGFBP2 GSK3B CCL5 0.72 0.806 1.526 0.821 0.722 0.92 PIGR ANGPT2 CCL5 0.72 0.806 1.526 0.824 0.72 0.929 PIGR LYVE1 CCL5 0.72 0.806 1.526 0.792 0.676 0.909 HSPA1A LGMN CCL5 0.72 0.806 1.526 0.786 0.668 0.903 ANGPT2 PDGFRβ PSPN 0.72 0.806 1.526 0.813 0.702 0.925 ANGPT2 FGF19 CCL5 0.72 0.806 1.526 0.816 0.698 0.933 PDGFRβ LYVE1 CCL5 0.72 0.806 1.526 0.802 0.691 0.914 LYVE1 FGF19 CCL5 0.72 0.806 1.526 0.793 0.67 0.916 SIGLEC7 PIGR HSPA1A 0.8 0.722 1.522 0.791 0.677 0.905 SIGLEC7 PIGR ANGPT2 0.8 0.722 1.522 0.809 0.702 0.916 SIGLEC7 HSPA1A PDGFRβ 0.8 0.722 1.522 0.784 0.666 0.903 SIGLEC7 LGMN IL12B- 0.8 0.722 1.522 0.868 0.78 0.956 IL23A SIGLEC7 LGMN PSPN 0.8 0.722 1.522 0.839 0.741 0.937 SIGLEC7 ANGPT2 PDGFRβ 0.8 0.722 1.522 0.819 0.709 0.929 SIGLEC7 PDGFRβ CCL5 0.8 0.722 1.522 0.83 0.727 0.933 IGFBP2 IGFBP1 HSPA1A 0.8 0.722 1.522 0.777 0.654 0.9 IGFBP2 PIGR GSK3B 0.8 0.722 1.522 0.812 0.709 0.916 IGFBP2 ANGPT2 GSK3B 0.8 0.722 1.522 0.798 0.689 0.907 IGFBP2 PDGFRβ FGF19 0.8 0.722 1.522 0.789 0.667 0.911 IGFBP2 LTA-LTB IL12B- 0.8 0.722 1.522 0.848 0.744 0.952 IL23A IGFBP2 IL12B- PSPN 0.8 0.722 1.522 0.81 0.7 0.92 IL23A IGFBP1 PIGR HSPA1A 0.8 0.722 1.522 0.802 0.688 0.916 IGFBP1 PIGR LTA-LTB 0.8 0.722 1.522 0.809 0.701 0.917 IGFBP1 ANGPT2 PSPN 0.8 0.722 1.522 0.804 0.692 0.917 PIGR GPC2 PDGFRβ 0.8 0.722 1.522 0.781 0.654 0.908 IGFBP2 ANGPT2 FGF19 0.68 0.833 1.513 0.77 0.644 0.896 SIGLEC7 IGFBP1 LYVE1 0.76 0.75 1.51 0.844 0.744 0.945 SIGLEC7 PIGR LTA-LTB 0.76 0.75 1.51 0.826 0.722 0.929 SIGLEC7 PIGR PSPN 0.76 0.75 1.51 0.794 0.682 0.907 SIGLEC7 HSPA1A LGMN 0.76 0.75 1.51 0.813 0.706 0.921 IGFBP2 IGFBP1 ANGPT2 0.76 0.75 1.51 0.789 0.67 0.908 IGFBP2 GPC2 PSPN 0.76 0.75 1.51 0.77 0.643 0.897 IGFBP2 LGMN ANGPT2 0.76 0.75 1.51 0.82 0.712 0.928 IGFBP2 GSK3B PSPN 0.76 0.75 1.51 0.787 0.681 0.892 IGFBP2 LYVE1 FGF19 0.76 0.75 1.51 0.764 0.639 0.89 IGFBP1 GPC2 LGMN 0.76 0.75 1.51 0.791 0.665 0.917 PIGR GPC2 LYVE1 0.76 0.75 1.51 0.77 0.641 0.899 HSPA1A LYVE1 CCL5 0.76 0.75 1.51 0.796 0.676 0.915 GPC2 LGMN PSPN 0.76 0.75 1.51 0.752 0.615 0.889 LGMN ANGPT2 PDGFRβ 0.76 0.75 1.51 0.822 0.715 0.929 LGMN LYVE1 FGF19 0.76 0.75 1.51 0.771 0.639 0.903 LGMN LYVE1 CCL5 0.76 0.75 1.51 0.817 0.703 0.931 LGMN LTA-LTB IL12B- 0.76 0.75 1.51 0.827 0.724 0.929 IL23A PDGFRβ CCL5 PSPN 0.76 0.75 1.51 0.794 0.679 0.91 SIGLEC7 LYVE1 IL12B- 0.84 0.667 1.507 0.84 0.733 0.947 IL23A IGFBP2 LGMN PSPN 0.84 0.667 1.507 0.838 0.737 0.939 IGFBP2 LTA-LTB PSPN 0.84 0.667 1.507 0.829 0.723 0.935 IGFBP1 HSPA1A IL12B- 0.84 0.667 1.507 0.759 0.636 0.882 IL23A PIGR PDGFRβ LTA-LTB 0.84 0.667 1.507 0.81 0.7 0.92 HSPA1A LTA-LTB CCL5 0.84 0.667 1.507 0.82 0.712 0.928 SIGLEC7 IGFBP2 HSPA1A 0.72 0.778 1.498 0.811 0.7 0.922 SIGLEC7 IGFBP1 PDGFRβ 0.72 0.778 1.498 0.826 0.719 0.932 SIGLEC7 IGFBP1 LTA-LTB 0.72 0.778 1.498 0.863 0.775 0.952 SIGLEC7 IGFBP1 PSPN 0.72 0.778 1.498 0.842 0.74 0.944 SIGLEC7 PIGR FGF19 0.72 0.778 1.498 0.804 0.688 0.921 SIGLEC7 GPC2 LTA-LTB 0.72 0.778 1.498 0.8 0.685 0.915 SIGLEC7 GPC2 PSPN 0.72 0.778 1.498 0.778 0.655 0.901 SIGLEC7 LGMN FGF19 0.72 0.778 1.498 0.834 0.724 0.945 SIGLEC7 PDGFRβ LTA-LTB 0.72 0.778 1.498 0.809 0.697 0.921 SIGLEC7 LYVE1 PSPN 0.72 0.778 1.498 0.804 0.69 0.919 SIGLEC7 FGF19 PSPN 0.72 0.778 1.498 0.799 0.679 0.918 IGFBP2 GPC2 PDGFRβ 0.72 0.778 1.498 0.786 0.663 0.908 IGFBP2 LGMN IL12B- 0.72 0.778 1.498 0.818 0.709 0.927 IL23A IGFBP2 LGMN FGF19 0.72 0.778 1.498 0.791 0.668 0.914 PIGR GPC2 LTA-LTB 0.72 0.778 1.498 0.768 0.637 0.898 PIGR LGMN CCL5 0.72 0.778 1.498 0.816 0.705 0.926 PIGR ANGPT2 LTA-LTB 0.72 0.778 1.498 0.799 0.683 0.915 PIGR ANGPT2 PSPN 0.72 0.778 1.498 0.786 0.672 0.899 GPC2 LGMN ANGPT2 0.72 0.778 1.498 0.766 0.64 0.891 GPC2 LTA-LTB PSPN 0.72 0.778 1.498 0.729 0.589 0.869 PDGFRβ LYVE1 FGF19 0.72 0.778 1.498 0.751 0.624 0.879 LTA-LTB IL12B- PSPN 0.72 0.778 1.498 0.82 0.715 0.925 IL23A SIGLEC7 PIGR GSK3B 0.8 0.694 1.494 0.809 0.71 0.908 SIGLEC7 HSPA1A CCL5 0.8 0.694 1.494 0.806 0.696 0.915 SIGLEC7 LGMN LYVE1 0.8 0.694 1.494 0.821 0.716 0.926 IGFBP2 IGFBP1 LTA-LTB 0.8 0.694 1.494 0.811 0.699 0.923 IGFBP2 PIGR LTA-LTB 0.8 0.694 1.494 0.804 0.688 0.921 IGFBP2 HSPA1A GSK3B 0.8 0.694 1.494 0.791 0.683 0.899 IGFBP2 ANGPT2 LTA-LTB 0.8 0.694 1.494 0.84 0.738 0.942 IGFBP1 HSPA1A PSPN 0.8 0.694 1.494 0.783 0.661 0.905 IGFBP1 PDGFRβ FGF19 0.8 0.694 1.494 0.767 0.641 0.892 PIGR HSPA1A GSK3B 0.8 0.694 1.494 0.803 0.701 0.905 PIGR HSPA1A LYVE1 0.8 0.694 1.494 0.784 0.664 0.904 HSPA1A IL12B- FGF19 0.8 0.694 1.494 0.72 0.586 0.854 IL23A HSPA1A CCL5 PSPN 0.8 0.694 1.494 0.771 0.653 0.889 LGMN IL12B- PSPN 0.8 0.694 1.494 0.791 0.679 0.903 IL23A LYVE1 LTA-LTB PSPN 0.8 0.694 1.494 0.796 0.679 0.912 SIGLEC7 IGFBP1 GSK3B 0.68 0.806 1.486 0.802 0.696 0.909 SIGLEC7 FGF19 CCL5 0.68 0.806 1.486 0.851 0.746 0.956 IGFBP2 ANGPT2 CCL5 0.68 0.806 1.486 0.821 0.715 0.927 IGFBP1 ANGPT2 FGF19 0.68 0.806 1.486 0.768 0.642 0.894 GPC2 PDGFRβ CCL5 0.68 0.806 1.486 0.749 0.618 0.879 SIGLEC7 IGFBP2 LYVE1 0.76 0.722 1.482 0.84 0.738 0.942 SIGLEC7 GPC2 LYVE1 0.76 0.722 1.482 0.797 0.675 0.918 SIGLEC7 PDGFRβ FGF19 0.76 0.722 1.482 0.812 0.697 0.927 IGFBP2 PIGR ANGPT2 0.76 0.722 1.482 0.798 0.682 0.914 IGFBP2 FGF19 PSPN 0.76 0.722 1.482 0.762 0.635 0.889 IGFBP1 PIGR CCL5 0.76 0.722 1.482 0.832 0.732 0.932 IGFBP1 PDGFRβ LYVE1 0.76 0.722 1.482 0.797 0.679 0.915 PIGR HSPA1A CCL5 0.76 0.722 1.482 0.793 0.677 0.91 PIGR LGMN IL12B- 0.76 0.722 1.482 0.778 0.661 0.894 IL23A PIGR IL12B- PSPN 0.76 0.722 1.482 0.784 0.671 0.898 IL23A PIGR CCL5 PSPN 0.76 0.722 1.482 0.79 0.677 0.903 HSPA1A LGMN LYVE1 0.76 0.722 1.482 0.79 0.671 0.909 LGMN ANGPT2 IL12B- 0.76 0.722 1.482 0.803 0.692 0.915 IL23A LGMN ANGPT2 PSPN 0.76 0.722 1.482 0.806 0.697 0.914 ANGPT2 LYVE1 PSPN 0.76 0.722 1.482 0.782 0.665 0.9 LYVE1 LTA-LTB FGF19 0.76 0.722 1.482 0.763 0.637 0.889 HSPA1A LGMN PSPN 0.84 0.639 1.479 0.766 0.642 0.889 IGFBP1 GSK3B LYVE1 0.64 0.833 1.473 0.787 0.671 0.902 GSK3B LTA-LTB IL12B- 0.64 0.833 1.473 0.786 0.679 0.892 IL23A SIGLEC7 IGFBP1 PIGR 0.72 0.75 1.47 0.856 0.763 0.948 SIGLEC7 GPC2 PDGFRβ 0.72 0.75 1.47 0.78 0.658 0.902 SIGLEC7 LGMN PDGFRβ 0.72 0.75 1.47 0.846 0.749 0.942 IGFBP2 PIGR GPC2 0.72 0.75 1.47 0.771 0.649 0.893 IGFBP2 ANGPT2 IL12B- 0.72 0.75 1.47 0.802 0.689 0.915 IL23A IGFBP1 LYVE1 CCL5 0.72 0.75 1.47 0.831 0.725 0.937 PIGR GPC2 LGMN 0.72 0.75 1.47 0.771 0.646 0.896 PIGR GPC2 IL12B- 0.72 0.75 1.47 0.761 0.635 0.887 IL23A PIGR GPC2 FGF19 0.72 0.75 1.47 0.741 0.609 0.873 PIGR GSK3B IL12B- 0.72 0.75 1.47 0.806 0.703 0.908 IL23A PIGR GSK3B CCL5 0.72 0.75 1.47 0.801 0.696 0.906 ANGPT2 LTA-LTB FGF19 0.72 0.75 1.47 0.788 0.666 0.91 ANGPT2 IL12B- FGF19 0.72 0.75 1.47 0.774 0.647 0.902 IL23A SIGLEC7 PIGR LGMN 0.8 0.667 1.467 0.833 0.728 0.938 IGFBP2 IGFBP1 LGMN 0.8 0.667 1.467 0.814 0.705 0.924 IGFBP2 HSPA1A LTA-LTB 0.8 0.667 1.467 0.813 0.703 0.924 IGFBP1 IL12B- CCL5 0.8 0.667 1.467 0.849 0.752 0.946 IL23A PIGR LGMN LTA-LTB 0.8 0.667 1.467 0.798 0.683 0.912 PIGR IL12B- FGF19 0.8 0.667 1.467 0.757 0.63 0.883 IL23A HSPA1A GPC2 PSPN 0.8 0.667 1.467 0.736 0.601 0.871 HSPA1A LTA-LTB PSPN 0.8 0.667 1.467 0.768 0.649 0.887 GPC2 IL12B- PSPN 0.8 0.667 1.467 0.753 0.625 0.882 IL23A PDGFRβ IL12B- FGF19 0.8 0.667 1.467 0.763 0.639 0.888 IL23A IGFBP1 PDGFRβ LTA-LTB 0.6 0.861 1.461 0.768 0.645 0.891 LGMN ANGPT2 CCL5 0.6 0.861 1.461 0.847 0.748 0.946 SIGLEC7 PIGR GPC2 0.68 0.778 1.458 0.798 0.682 0.914 SIGLEC7 HSPA1A GPC2 0.68 0.778 1.458 0.78 0.658 0.902 SIGLEC7 GSK3B LYVE1 0.68 0.778 1.458 0.81 0.709 0.911 SIGLEC7 PDGFRβ PSPN 0.68 0.778 1.458 0.807 0.692 0.921 IGFBP2 GSK3B LYVE1 0.68 0.778 1.458 0.812 0.713 0.912 IGFBP1 GPC2 PSPN 0.68 0.778 1.458 0.762 0.629 0.896 PIGR GSK3B FGF19 0.68 0.778 1.458 0.769 0.649 0.889 GPC2 LYVE1 PSPN 0.68 0.778 1.458 0.74 0.605 0.875 GPC2 CCL5 PSPN 0.68 0.778 1.458 0.756 0.621 0.89 LGMN GSK3B LTA-LTB 0.68 0.778 1.458 0.782 0.67 0.894 LGMN PDGFRβ CCL5 0.68 0.778 1.458 0.837 0.734 0.939 LGMN IL12B- CCL5 0.68 0.778 1.458 0.824 0.715 0.933 IL23A PDGFRβ LTA-LTB FGF19 0.68 0.778 1.458 0.778 0.654 0.902 SIGLEC7 HSPA1A LYVE1 0.76 0.694 1.454 0.772 0.649 0.895 SIGLEC7 LGMN LTA-LTB 0.76 0.694 1.454 0.859 0.767 0.951 SIGLEC7 ANGPT2 LYVE1 0.76 0.694 1.454 0.789 0.673 0.905 IGFBP2 IGFBP1 IL12B- 0.76 0.694 1.454 0.786 0.669 0.902 IL23A IGFBP2 IGFBP1 PSPN 0.76 0.694 1.454 0.786 0.662 0.909 IGFBP2 HSPA1A ANGPT2 0.76 0.694 1.454 0.749 0.624 0.874 IGFBP1 PIGR IL12B- 0.76 0.694 1.454 0.81 0.701 0.919 IL23A IGFBP1 PIGR PSPN 0.76 0.694 1.454 0.784 0.669 0.9 IGFBP1 HSPA1A ANGPT2 0.76 0.694 1.454 0.773 0.651 0.895 IGFBP1 HSPA1A PDGFRβ 0.76 0.694 1.454 0.772 0.65 0.894 IGFBP1 LGMN PDGFRβ 0.76 0.694 1.454 0.831 0.724 0.939 IGFBP1 ANGPT2 LTA-LTB 0.76 0.694 1.454 0.819 0.712 0.925 PIGR LTA-LTB IL12B- 0.76 0.694 1.454 0.806 0.695 0.916 IL23A HSPA1A GPC2 IL12B- 0.76 0.694 1.454 0.743 0.614 0.873 IL23A HSPA1A LGMN LTA-LTB 0.76 0.694 1.454 0.801 0.687 0.915 HSPA1A LGMN FGF19 0.76 0.694 1.454 0.751 0.62 0.883 HSPA1A PDGFRβ LYVE1 0.76 0.694 1.454 0.749 0.623 0.875 LYVE1 IL12B- FGF19 0.76 0.694 1.454 0.761 0.631 0.891 IL23A LYVE1 IL12B- PSPN 0.76 0.694 1.454 0.777 0.654 0.899 IL23A LTA-LTB FGF19 PSPN 0.76 0.694 1.454 0.771 0.641 0.901 SIGLEC7 IGFBP1 FGF19 0.64 0.806 1.446 0.832 0.724 0.941 SIGLEC7 GPC2 CCL5 0.64 0.806 1.446 0.811 0.7 0.922 SIGLEC7 LGMN GSK3B 0.64 0.806 1.446 0.828 0.732 0.924 SIGLEC7 GSK3B FGF19 0.64 0.806 1.446 0.79 0.675 0.905 IGFBP2 PIGR IL12B- 0.64 0.806 1.446 0.768 0.647 0.888 IL23A IGFBP2 GPC2 GSK3B 0.64 0.806 1.446 0.757 0.639 0.874 IGFBP1 GPC2 CCL5 0.64 0.806 1.446 0.78 0.658 0.902 PIGR GPC2 CCL5 0.64 0.806 1.446 0.778 0.654 0.901 PIGR IL12B- CCL5 0.64 0.806 1.446 0.831 0.73 0.933 IL23A GPC2 LGMN CCL5 0.64 0.806 1.446 0.763 0.633 0.893 SIGLEC7 PIGR LYVE1 0.72 0.722 1.442 0.803 0.695 0.911 SIGLEC7 ANGPT2 LTA-LTB 0.72 0.722 1.442 0.829 0.726 0.932 SIGLEC7 PDGFRβ LYVE1 0.72 0.722 1.442 0.79 0.674 0.906 IGFBP2 PIGR LYVE1 0.72 0.722 1.442 0.796 0.682 0.909 IGFBP2 PIGR FGF19 0.72 0.722 1.442 0.752 0.624 0.88 IGFBP2 PIGR CCL5 0.72 0.722 1.442 0.812 0.704 0.921 IGFBP2 HSPA1A GPC2 0.72 0.722 1.442 0.773 0.65 0.897 IGFBP2 HSPA1A LYVE1 0.72 0.722 1.442 0.76 0.636 0.884 IGFBP2 LTA-LTB FGF19 0.72 0.722 1.442 0.786 0.664 0.907 IGFBP1 IL12B- FGF19 0.72 0.722 1.442 0.76 0.634 0.886 IL23A IGFBP1 FGF19 CCL5 0.72 0.722 1.442 0.794 0.675 0.914 PIGR HSPA1A GPC2 0.72 0.722 1.442 0.781 0.66 0.902 PIGR LGMN ANGPT2 0.72 0.722 1.442 0.803 0.695 0.912 PIGR LGMN FGF19 0.72 0.722 1.442 0.79 0.663 0.917 HSPA1A GPC2 CCL5 0.72 0.722 1.442 0.759 0.632 0.886 HSPA1A ANGPT2 CCL5 0.72 0.722 1.442 0.757 0.633 0.88 HSPA1A GSK3B LTA-LTB 0.72 0.722 1.442 0.794 0.692 0.897 HSPA1A GSK3B CCL5 0.72 0.722 1.442 0.803 0.703 0.904 HSPA1A LYVE1 FGF19 0.72 0.722 1.442 0.736 0.602 0.869 GPC2 ANGPT2 LYVE1 0.72 0.722 1.442 0.746 0.612 0.88 GPC2 ANGPT2 IL12B- 0.72 0.722 1.442 0.754 0.628 0.881 IL23A GPC2 LYVE1 IL12B- 0.72 0.722 1.442 0.742 0.608 0.876 IL23A GPC2 FGF19 PSPN 0.72 0.722 1.442 0.758 0.624 0.892 LGMN PDGFRβ LTA-LTB 0.72 0.722 1.442 0.804 0.688 0.921 ANGPT2 FGF19 PSPN 0.72 0.722 1.442 0.778 0.652 0.903 PDGFRβ LTA-LTB CCL5 0.72 0.722 1.442 0.822 0.718 0.927 PDGFRβ FGF19 PSPN 0.72 0.722 1.442 0.762 0.636 0.889 LYVE1 FGF19 PSPN 0.72 0.722 1.442 0.743 0.614 0.873 HSPA1A PDGFRβ CCL5 0.8 0.639 1.439 0.784 0.671 0.897 HSPA1A LYVE1 IL12B- 0.8 0.639 1.439 0.786 0.664 0.907 IL23A LGMN GSK3B FGF19 0.6 0.833 1.433 0.774 0.653 0.896 GSK3B LTA-LTB FGF19 0.6 0.833 1.433 0.778 0.659 0.897 PDGFRβ LTA-LTB IL12B- 0.6 0.833 1.433 0.741 0.612 0.87 IL23A SIGLEC7 HSPA1A ANGPT2 0.68 0.75 1.43 0.767 0.649 0.885 SIGLEC7 ANGPT2 GSK3B 0.68 0.75 1.43 0.802 0.699 0.905 SIGLEC7 GSK3B CCL5 0.68 0.75 1.43 0.83 0.734 0.926 IGFBP2 GPC2 LTA-LTB 0.68 0.75 1.43 0.788 0.668 0.908 IGFBP1 GPC2 ANGPT2 0.68 0.75 1.43 0.767 0.642 0.892 IGFBP1 GPC2 LTA-LTB 0.68 0.75 1.43 0.76 0.633 0.887 PIGR LGMN GSK3B 0.68 0.75 1.43 0.801 0.695 0.907 PIGR LTA-LTB FGF19 0.68 0.75 1.43 0.766 0.638 0.893 HSPA1A GPC2 ANGPT2 0.68 0.75 1.43 0.744 0.614 0.875 HSPA1A GPC2 FGF19 0.68 0.75 1.43 0.727 0.591 0.862 HSPA1A LGMN GSK3B 0.68 0.75 1.43 0.788 0.682 0.893 HSPA1A GSK3B FGF19 0.68 0.75 1.43 0.758 0.638 0.877 HSPA1A FGF19 CCL5 0.68 0.75 1.43 0.753 0.626 0.881 GPC2 LYVE1 LTA-LTB 0.68 0.75 1.43 0.76 0.631 0.889 IGFBP2 IGFBP1 LYVE1 0.76 0.667 1.427 0.784 0.666 0.902 IGFBP2 PIGR LGMN 0.76 0.667 1.427 0.814 0.704 0.925 IGFBP2 HSPA1A PSPN 0.76 0.667 1.427 0.776 0.659 0.892 IGFBP1 LGMN IL12B- 0.76 0.667 1.427 0.802 0.694 0.911 IL23A IGFBP1 LGMN FGF19 0.76 0.667 1.427 0.786 0.663 0.908 IGFBP1 LGMN PSPN 0.76 0.667 1.427 0.81 0.699 0.921 PIGR LGMN LYVE1 0.76 0.667 1.427 0.787 0.669 0.904 PIGR LYVE1 FGF19 0.76 0.667 1.427 0.76 0.632 0.888 PIGR LTA-LTB PSPN 0.76 0.667 1.427 0.773 0.65 0.897 HSPA1A ANGPT2 PDGFRβ 0.76 0.667 1.427 0.758 0.636 0.88 HSPA1A PDGFRβ FGF19 0.76 0.667 1.427 0.743 0.615 0.872 ANGPT2 LYVE1 IL12B- 0.76 0.667 1.427 0.763 0.64 0.887 IL23A IL12B- FGF19 PSPN 0.76 0.667 1.427 0.79 0.665 0.915 IL23A ANGPT2 LTA-LTB IL12B- 0.84 0.583 1.423 0.833 0.734 0.933 IL23A SIGLEC7 IGFBP1 GPC2 0.64 0.778 1.418 0.82 0.708 0.932 SIGLEC7 GPC2 FGF19 0.64 0.778 1.418 0.788 0.667 0.908 IGFBP2 GPC2 LYVE1 0.64 0.778 1.418 0.769 0.644 0.894 IGFBP2 GPC2 CCL5 0.64 0.778 1.418 0.79 0.67 0.91 IGFBP1 GPC2 LYVE1 0.64 0.778 1.418 0.768 0.641 0.895 PIGR ANGPT2 FGF19 0.64 0.778 1.418 0.777 0.651 0.903 HSPA1A GPC2 LGMN 0.64 0.778 1.418 0.761 0.633 0.889 HSPA1A ANGPT2 FGF19 0.64 0.778 1.418 0.746 0.614 0.877 GPC2 LGMN FGF19 0.64 0.778 1.418 0.75 0.617 0.883 GPC2 ANGPT2 FGF19 0.64 0.778 1.418 0.737 0.605 0.868 GPC2 ANGPT2 CCL5 0.64 0.778 1.418 0.773 0.651 0.896 GPC2 LYVE1 CCL5 0.64 0.778 1.418 0.758 0.625 0.89 GPC2 IL12B- CCL5 0.64 0.778 1.418 0.74 0.607 0.873 IL23A LGMN GSK3B PDGFRβ 0.64 0.778 1.418 0.761 0.65 0.873 GSK3B LTA-LTB CCL5 0.64 0.778 1.418 0.819 0.722 0.916 PDGFRβ LTA-LTB PSPN 0.64 0.778 1.418 0.739 0.612 0.866 SIGLEC7 HSPA1A GSK3B 0.72 0.694 1.414 0.796 0.69 0.901 SIGLEC7 GPC2 IL12B- 0.72 0.694 1.414 0.818 0.706 0.929 IL23A SIGLEC7 LYVE1 FGF19 0.72 0.694 1.414 0.803 0.686 0.921 IGFBP2 HSPA1A LGMN 0.72 0.694 1.414 0.783 0.667 0.9 IGFBP2 LYVE1 PSPN 0.72 0.694 1.414 0.791 0.673 0.91 IGFBP1 IL12B- PSPN 0.72 0.694 1.414 0.822 0.717 0.927 IL23A PIGR ANGPT2 GSK3B 0.72 0.694 1.414 0.796 0.69 0.901 PIGR LYVE1 IL12B- 0.72 0.694 1.414 0.778 0.658 0.898 IL23A GPC2 ANGPT2 PSPN 0.72 0.694 1.414 0.756 0.628 0.884 GPC2 LTA-LTB IL12B- 0.72 0.694 1.414 0.767 0.641 0.892 IL23A LGMN PDGFRβ PSPN 0.72 0.694 1.414 0.788 0.672 0.903 LGMN LTA-LTB PSPN 0.72 0.694 1.414 0.788 0.668 0.908 LGMN IL12B- FGF19 0.72 0.694 1.414 0.784 0.659 0.909 IL23A ANGPT2 LYVE1 CCL5 0.72 0.694 1.414 0.814 0.7 0.929 PIGR PDGFRβ PSPN 0.8 0.611 1.411 0.789 0.677 0.901 GPC2 GSK3B LYVE1 0.6 0.806 1.406 0.749 0.627 0.871 LGMN GSK3B IL12B- 0.6 0.806 1.406 0.786 0.681 0.891 IL23A LGMN GSK3B CCL5 0.6 0.806 1.406 0.803 0.701 0.906 ANGPT2 GSK3B FGF19 0.6 0.806 1.406 0.77 0.652 0.888 IGFBP2 GPC2 ANGPT2 0.68 0.722 1.402 0.768 0.645 0.89 IGFBP1 LYVE1 FGF19 0.68 0.722 1.402 0.758 0.632 0.883 PIGR GPC2 GSK3B 0.68 0.722 1.402 0.756 0.634 0.877 PIGR GPC2 PSPN 0.68 0.722 1.402 0.739 0.604 0.874 PIGR ANGPT2 IL12B- 0.68 0.722 1.402 0.788 0.675 0.901 IL23A PIGR GSK3B LYVE1 0.68 0.722 1.402 0.801 0.696 0.906 HSPA1A GSK3B IL12B- 0.68 0.722 1.402 0.783 0.678 0.889 IL23A GPC2 LGMN PDGFRβ 0.68 0.722 1.402 0.749 0.614 0.884 GPC2 LGMN IL12B- 0.68 0.722 1.402 0.739 0.609 0.869 IL23A GPC2 ANGPT2 PDGFRβ 0.68 0.722 1.402 0.766 0.638 0.893 GPC2 ANGPT2 LTA-LTB 0.68 0.722 1.402 0.774 0.654 0.895 GPC2 IL12B- FGF19 0.68 0.722 1.402 0.73 0.599 0.861 IL23A LGMN ANGPT2 LTA-LTB 0.68 0.722 1.402 0.827 0.725 0.928 IGFBP2 PIGR PSPN 0.76 0.639 1.399 0.774 0.657 0.891 PIGR LGMN PSPN 0.76 0.639 1.399 0.769 0.647 0.891 PIGR LYVE1 PSPN 0.76 0.639 1.399 0.78 0.663 0.897 HSPA1A LGMN PDGFRβ 0.76 0.639 1.399 0.787 0.672 0.901 HSPA1A PDGFRβ IL12B- 0.76 0.639 1.399 0.754 0.634 0.874 IL23A HSPA1A IL12B- PSPN 0.76 0.639 1.399 0.739 0.616 0.862 IL23A LGMN PDGFRβ IL12B- 0.76 0.639 1.399 0.813 0.707 0.919 IL23A PDGFRβ IL12B- PSPN 0.76 0.639 1.399 0.772 0.656 0.888 IL23A IGFBP1 GSK3B CCL5 0.56 0.833 1.393 0.788 0.677 0.898 IGFBP1 GSK3B PSPN 0.56 0.833 1.393 0.717 0.591 0.842 GSK3B LYVE1 IL12B- 0.56 0.833 1.393 0.782 0.677 0.888 IL23A GSK3B IL12B- CCL5 0.56 0.833 1.393 0.83 0.734 0.926 IL23A GSK3B FGF19 CCL5 0.56 0.833 1.393 0.779 0.661 0.897 SIGLEC7 HSPA1A FGF19 0.64 0.75 1.39 0.78 0.658 0.902 SIGLEC7 GPC2 ANGPT2 0.64 0.75 1.39 0.796 0.683 0.908 SIGLEC7 ANGPT2 FGF19 0.64 0.75 1.39 0.821 0.71 0.932 IGFBP2 HSPA1A CCL5 0.64 0.75 1.39 0.809 0.702 0.916 IGFBP2 GSK3B FGF19 0.64 0.75 1.39 0.766 0.649 0.882 PIGR GPC2 ANGPT2 0.64 0.75 1.39 0.772 0.651 0.893 GPC2 FGF19 CCL5 0.64 0.75 1.39 0.753 0.621 0.886 ANGPT2 PDGFRβ LTA-LTB 0.64 0.75 1.39 0.789 0.676 0.902 GSK3B CCL5 PSPN 0.64 0.75 1.39 0.792 0.687 0.898 SIGLEC7 LGMN ANGPT2 0.72 0.667 1.387 0.832 0.733 0.931 SIGLEC7 ANGPT2 IL12B- 0.72 0.667 1.387 0.833 0.732 0.935 IL23A IGFBP2 ANGPT2 LYVE1 0.72 0.667 1.387 0.789 0.67 0.908 IGFBP1 PIGR ANGPT2 0.72 0.667 1.387 0.813 0.708 0.918 IGFBP1 PIGR LYVE1 0.72 0.667 1.387 0.809 0.703 0.915 IGFBP1 GPC2 IL12B- 0.72 0.667 1.387 0.761 0.637 0.886 IL23A IGFBP1 LTA-LTB FGF19 0.72 0.667 1.387 0.766 0.64 0.891 PIGR HSPA1A ANGPT2 0.72 0.667 1.387 0.764 0.641 0.888 PIGR HSPA1A FGF19 0.72 0.667 1.387 0.741 0.611 0.871 HSPA1A LGMN ANGPT2 0.72 0.667 1.387 0.749 0.625 0.873 HSPA1A LGMN IL12B- 0.72 0.667 1.387 0.741 0.616 0.866 IL23A HSPA1A ANGPT2 LYVE1 0.72 0.667 1.387 0.749 0.621 0.877 HSPA1A ANGPT2 LTA-LTB 0.72 0.667 1.387 0.769 0.651 0.886 HSPA1A ANGPT2 IL12B- 0.72 0.667 1.387 0.747 0.619 0.874 IL23A HSPA1A ANGPT2 PSPN 0.72 0.667 1.387 0.739 0.615 0.863 HSPA1A IL12B- CCL5 0.72 0.667 1.387 0.766 0.644 0.888 IL23A LGMN LYVE1 PSPN 0.72 0.667 1.387 0.778 0.661 0.894 ANGPT2 LYVE1 LTA-LTB 0.72 0.667 1.387 0.777 0.662 0.892 PIGR HSPA1A PSPN 0.8 0.583 1.383 0.758 0.635 0.881 SIGLEC7 GSK3B PDGFRβ 0.6 0.778 1.378 0.792 0.687 0.897 SIGLEC7 GSK3B IL12B- 0.6 0.778 1.378 0.831 0.735 0.927 IL23A HSPA1A GPC2 GSK3B 0.6 0.778 1.378 0.751 0.632 0.871 GPC2 GSK3B FGF19 0.6 0.778 1.378 0.731 0.603 0.859 LGMN GSK3B LYVE1 0.6 0.778 1.378 0.79 0.685 0.895 IGFBP2 LYVE1 IL12B- 0.68 0.694 1.374 0.781 0.66 0.903 IL23A GPC2 PDGFRβ LYVE1 0.68 0.694 1.374 0.721 0.587 0.855 GPC2 LTA-LTB FGF19 0.68 0.694 1.374 0.76 0.632 0.888 IL12B- FGF19 CCL5 0.68 0.694 1.374 0.81 0.691 0.929 IL23A IGFBP1 HSPA1A FGF19 0.76 0.611 1.371 0.76 0.631 0.889 LGMN LYVE1 LTA-LTB 0.76 0.611 1.371 0.807 0.697 0.916 ANGPT2 PDGFRβ LYVE1 0.76 0.611 1.371 0.754 0.634 0.875 LYVE1 LTA-LTB IL12B- 0.76 0.611 1.371 0.797 0.684 0.909 IL23A GSK3B LYVE1 LTA-LTB 0.56 0.806 1.366 0.781 0.672 0.89 GSK3B FGF19 PSPN 0.56 0.806 1.366 0.741 0.618 0.865 IGFBP2 GSK3B PDGFRβ 0.64 0.722 1.362 0.787 0.679 0.895 IGFBP1 PIGR GSK3B 0.64 0.722 1.362 0.769 0.659 0.879 PIGR GSK3B LTA-LTB 0.64 0.722 1.362 0.797 0.688 0.906 PIGR GSK3B PSPN 0.64 0.722 1.362 0.766 0.655 0.876 HSPA1A GSK3B LYVE1 0.64 0.722 1.362 0.798 0.694 0.902 IGFBP2 HSPA1A IL12B- 0.72 0.639 1.359 0.756 0.635 0.876 IL23A PIGR HSPA1A IL12B- 0.72 0.639 1.359 0.758 0.637 0.878 IL23A HSPA1A FGF19 PSPN 0.72 0.639 1.359 0.733 0.6 0.866 IGFBP1 LGMN GSK3B 0.52 0.833 1.353 0.778 0.667 0.889 IGFBP1 GSK3B LTA-LTB 0.52 0.833 1.353 0.781 0.671 0.892 SIGLEC7 GSK3B LTA-LTB 0.6 0.75 1.35 0.818 0.717 0.919 SIGLEC7 GSK3B PSPN 0.6 0.75 1.35 0.802 0.7 0.904 IGFBP2 IL12B- FGF19 0.6 0.75 1.35 0.769 0.645 0.893 IL23A GPC2 GSK3B PSPN 0.6 0.75 1.35 0.726 0.597 0.854 GPC2 PDGFRβ PSPN 0.6 0.75 1.35 0.704 0.566 0.843 LGMN ANGPT2 GSK3B 0.6 0.75 1.35 0.787 0.681 0.893 LGMN GSK3B PSPN 0.6 0.75 1.35 0.746 0.626 0.865 GSK3B PDGFRβ LTA-LTB 0.6 0.75 1.35 0.749 0.631 0.866 IGFBP2 IGFBP1 FGF19 0.68 0.667 1.347 0.753 0.626 0.88 IGFBP1 LTA-LTB PSPN 0.68 0.667 1.347 0.783 0.668 0.899 IGFBP1 FGF19 PSPN 0.68 0.667 1.347 0.76 0.631 0.889 PIGR FGF19 PSPN 0.68 0.667 1.347 0.746 0.615 0.877 HSPA1A PDGFRβ LTA-LTB 0.68 0.667 1.347 0.772 0.652 0.893 GPC2 PDGFRβ FGF19 0.68 0.667 1.347 0.739 0.607 0.871 ANGPT2 GSK3B IL12B- 0.48 0.861 1.341 0.784 0.68 0.889 IL23A IGFBP1 GPC2 GSK3B 0.56 0.778 1.338 0.722 0.596 0.849 GPC2 LGMN GSK3B 0.56 0.778 1.338 0.732 0.603 0.862 GPC2 GSK3B CCL5 0.56 0.778 1.338 0.728 0.599 0.857 GSK3B LYVE1 CCL5 0.56 0.778 1.338 0.823 0.724 0.923 GSK3B LYVE1 PSPN 0.56 0.778 1.338 0.774 0.665 0.884 IGFBP2 IGFBP1 PIGR 0.64 0.694 1.334 0.774 0.658 0.891 IGFBP2 GPC2 IL12B- 0.64 0.694 1.334 0.744 0.618 0.871 IL23A PIGR GSK3B PDGFRβ 0.64 0.694 1.334 0.784 0.681 0.888 HSPA1A ANGPT2 GSK3B 0.64 0.694 1.334 0.764 0.654 0.875 HSPA1A GSK3B PSPN 0.64 0.694 1.334 0.75 0.635 0.865 GPC2 PDGFRβ IL12B- 0.64 0.694 1.334 0.726 0.595 0.856 IL23A PDGFRβ LYVE1 LTA-LTB 0.64 0.694 1.334 0.757 0.633 0.881 PDGFRβ LYVE1 PSPN 0.64 0.694 1.334 0.759 0.634 0.884 HSPA1A PDGFRβ PSPN 0.72 0.611 1.331 0.737 0.612 0.862 LGMN PDGFRβ LYVE1 0.72 0.611 1.331 0.8 0.69 0.91 GSK3B IL12B- PSPN 0.52 0.806 1.326 0.761 0.651 0.871 IL23A IGFBP2 GPC2 FGF19 0.6 0.722 1.322 0.718 0.585 0.85 IGFBP1 HSPA1A GSK3B 0.6 0.722 1.322 0.772 0.658 0.886 IGFBP1 GPC2 FGF19 0.6 0.722 1.322 0.734 0.603 0.866 PIGR ANGPT2 LYVE1 0.68 0.639 1.319 0.777 0.659 0.894 PDGFRβ LYVE1 IL12B- 0.68 0.639 1.319 0.74 0.615 0.865 IL23A SIGLEC7 GPC2 GSK3B 0.56 0.75 1.31 0.766 0.648 0.883 IGFBP1 ANGPT2 GSK3B 0.56 0.75 1.31 0.757 0.64 0.873 ANGPT2 GSK3B LTA-LTB 0.56 0.75 1.31 0.772 0.663 0.881 GSK3B PDGFRβ LYVE1 0.56 0.75 1.31 0.75 0.636 0.864 GSK3B PDGFRβ FGF19 0.56 0.75 1.31 0.739 0.617 0.861 HSPA1A GSK3B PDGFRβ 0.64 0.667 1.307 0.753 0.641 0.865 GPC2 GSK3B PDGFRβ 0.52 0.778 1.298 0.708 0.58 0.835 GPC2 GSK3B LTA-LTB 0.52 0.778 1.298 0.736 0.609 0.862 GPC2 PDGFRβ LTA-LTB 0.52 0.778 1.298 0.707 0.569 0.844 ANGPT2 GSK3B CCL5 0.52 0.778 1.298 0.818 0.719 0.916 ANGPT2 GSK3B PSPN 0.52 0.778 1.298 0.762 0.653 0.872 GSK3B LYVE1 FGF19 0.52 0.778 1.298 0.772 0.655 0.889 GSK3B IL12B- FGF19 0.52 0.778 1.298 0.75 0.63 0.87 IL23A IGFBP1 GPC2 PDGFRβ 0.6 0.694 1.294 0.739 0.607 0.871 IGFBP1 LYVE1 PSPN 0.6 0.694 1.294 0.773 0.654 0.893 GPC2 LYVE1 FGF19 0.6 0.694 1.294 0.714 0.576 0.853 IGFBP1 GSK3B PDGFRβ 0.48 0.806 1.286 0.742 0.62 0.864 IGFBP1 GSK3B IL12B- 0.48 0.806 1.286 0.761 0.647 0.875 IL23A GPC2 ANGPT2 GSK3B 0.48 0.806 1.286 0.738 0.617 0.859 GPC2 GSK3B IL12B- 0.48 0.806 1.286 0.732 0.609 0.855 IL23A GSK3B PDGFRβ IL12B- 0.48 0.806 1.286 0.744 0.63 0.859 IL23A ANGPT2 GSK3B LYVE1 0.56 0.722 1.282 0.773 0.666 0.881 IGFBP1 GSK3B FGF19 0.52 0.75 1.27 0.728 0.604 0.852 GSK3B LTA-LTB PSPN 0.52 0.75 1.27 0.76 0.648 0.872 IGFBP1 PDGFRβ PSPN 0.6 0.667 1.267 0.746 0.617 0.874 HSPA1A GPC2 PDGFRβ 0.6 0.667 1.267 0.734 0.606 0.863 IGFBP1 PIGR FGF19 0.68 0.583 1.263 0.753 0.626 0.88 ANGPT2 GSK3B PDGFRβ 0.48 0.778 1.258 0.779 0.668 0.89 IGFBP2 HSPA1A FGF19 0.6 0.639 1.239 0.726 0.595 0.856 GSK3B PDGFRβ PSPN 0.48 0.75 1.23 0.746 0.63 0.861 GSK3B PDGFRβ CCL5 0.48 0.722 1.202 0.79 0.684 0.896

The foregoing embodiments and examples are intended only as examples. No particular embodiment, example, or element of a particular embodiment or example is to be construed as a critical, required, or essential element or feature of any of the claims. Various alterations, modifications, substitutions, and other variations can be made to the disclosed embodiments without departing from the scope of the present application, which is defined by the appended claims. The specification, including the figures and examples, is to be regarded in an illustrative manner, rather than a restrictive one, and all such modifications and substitutions are intended to be included within the scope of the application. Steps recited in any of the method or process claims may be executed in any feasible order and are not limited to an order presented in any of the embodiments, the examples, or the claims. Further, in any of the aforementioned methods, one or more specifically listed biomarkers can be specifically excluded either as an individual biomarker or as a biomarker from any panel. 

The invention claimed is:
 1. A method of predicting whether lung function will decline in a subject with chronic obstructive pulmonary disease (COPD) or at risk of developing COPD, comprising detecting the level of each biomarker protein of a set of biomarker proteins comprising SIGLEC7, IGFBP1, and CCL5 in a sample from the subject, wherein the detecting comprises: (i) contacting proteins of the sample with a set of protein binding reagents, wherein each protein binding reagent of the set of protein binding reagents specifically binds to a different biomarker protein being detected; (ii) separating protein binding reagents that are bound to biomarker protein from protein binding reagents that are not bound to biomarker protein; and (iii) detecting the separated protein binding reagents; thereby detecting the level of each of the set of biomarker proteins in the sample.
 2. The method of claim 1, wherein each protein binding reagent is an antibody or an aptamer.
 3. The method of claim 1, wherein the set of biomarker proteins further comprises at least one biomarker protein selected from GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN.
 4. The method of claim 1, wherein the set of biomarker proteins further comprises PSPN.
 5. The method of claim 1, wherein the set of biomarker proteins further comprises LTA LTB.
 6. The method of claim 1, wherein the set of biomarker proteins further comprises GPC2.
 7. The method of claim 1, wherein the set of biomarker proteins further comprises GPC2, LYVE1, PSPN, CCDC80, and IGFBP-7.
 8. The method of claim 1, wherein the set of biomarker proteins further comprises IGFBP2, LTA LTB, PSPN, GSK3β, and IGFBP1.
 9. The method of claim 1, wherein the set of biomarker proteins further comprises at least three biomarker proteins selected from PSPN, LTA LTB, GSK3β, THPO, GPC2, ANGPT2, PIGR, FST, HS6ST1, LGALS3, IL10, and LGMN.
 10. The method of claim 1, wherein the set of biomarker proteins further comprises IGFBP2, LTA LTB, PSPN, and GSK3β.
 11. The method of claim 1, wherein at least one protein binding reagent is a slow off-rate aptamer.
 12. The method of claim 11, wherein each slow off-rate aptamer binds to its target protein with an off rate (t_(1/2)) of ≧30 minutes.
 13. The method of claim 11, wherein at least one protein binding reagent is a slow off-rate aptamer comprising at least one nucleotide with a modification.
 14. The method of claim 11, wherein the sample is a blood sample.
 15. The method of claim 14, wherein the sample is selected from a serum sample and a plasma sample.
 16. The method of claim 1, wherein the method further comprises treating the subject for COPD.
 17. The method of claim 16, wherein treating the subject for COPD comprises at least one treatment selected from a smoking cessation program, a long-acting bronchodilator, an inhaled corticosteroid, and a systemic corticosteroid.
 18. The method of claim 17, wherein treating the subject for COPD comprises at least one of a therapeutic agent that aids in smoking cessation; behavior modification therapy; and/or at least one treatment selected from a β2-agonist, an anticholinergic, beclomethasone, budesonide, flunisolide, fluticasone, mometasone, triamcinolone, methylprednisolone, prednisolone, and prednisone.
 19. The method of claim 1, wherein at least one protein binding reagent is an aptamer and detecting the separated protein binding reagents comprises at least one method selected from amplification and hybridization.
 20. A method of predicting whether lung function will decline in a subject with chronic obstructive pulmonary disease (COPD) or at risk of developing COPD, comprising detecting the level of each biomarker protein of a set of biomarker proteins comprising SIGLEC7 and IGFBP1 in a sample from the subject, wherein the detecting comprises: (i) contacting proteins of the sample with a set of protein binding reagents, wherein each protein binding reagent of the set of protein binding reagents specifically binds to a different biomarker protein being detected; (ii) separating protein binding reagents that are bound to biomarker protein from protein binding reagents that are not bound to biomarker protein; and (iii) detecting the separated protein binding reagents; thereby detecting the level of each of the set of biomarker proteins in the sample.
 21. The method of claim 20, wherein each protein binding reagent is an antibody or an aptamer.
 22. The method of claim 20, wherein the set of biomarker proteins further comprises at least one biomarker protein selected from GPC2, LYVE1, PSPN, CCDC80, IGFBP-7, PIGR, ANGPT2, GSK3β, LTA LTB, FGF19, THPO, MST1R, CHST15, COL18A1, IGFBP2, HSPA1A, SLPI, CXCL5, SOD2, CCL22, FGFR2, IGFBP4, FST, HS6ST1, LGALS3, IL10, and LGMN.
 23. A kit comprising a set of aptamers, wherein the set of aptamers comprises an aptamer that specifically binds to SIGLEC7, an aptamer that specifically binds to IGFBP1, and an aptamer that specifically binds to CCL5. 